Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

RELATED APPLICATIONS

This application is a continuation of, and claims priority under 35 USC§120 to, U.S. application Ser. No. 10/797,366 filed Mar. 9, 2004, whichis a continuation of, and claims priority under 35 USC §120 to, U.S.application Ser. No. 09/665,350 filed Sep. 18, 2000 now abandoned, whichis a continuation of, and claims priority under 35 USC §120 to, PCTApplication PCT/US00/04414 filed Feb. 22, 2000, which is acontinuation-in-part of, and claims priority under 35 USC §120 to, PCTApplication PCT/US98/19330 filed Sep. 16, 1998, which is acontinuation-in-part of, and claims priority under 35 USC §120 to, PCTApplication PCT/US98/18824 filed Sep. 10, 1998, which claims priorityunder 35 USC §119 to U.S. Provisional Application 60/062,285 filed Oct.17, 1997, the entire disclosures of which are hereby expresslyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108–7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immunoadhesins, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

Growth factors are molecular signals or mediators that enhance cellgrowth or proliferation, alone or in concert, by binding to specificcell surface receptors. However, there are other cellular reactions thanonly growth upon expression to growth factors. As a result, growthfactors are better characterized as multifunctional and potent cellularregulators. Their biological effects include proliferation, chemotaxisand stimulation of extracellular matrix production. Growth factors canhave both stimulatory and inhibitory effects. For example, transforminggrowth factor (TGF-β) is highly pleiotropic and can stimulateproliferation in some cells, especially connective tissue, while being apotent inhibitor of proliferation in others, such as lymphocytes andepithelial cells.

The physiological effect of growth stimulation or inhibition by growthfactors depends upon the state of development and differentiation of thetarget tissue. The mechanism of local cellular regulation by classicalendocrine molecules involves comprehends autocrine (same cell),juxtacrine (neighbor cell), and paracrine (adjacent cells) pathways.Peptide growth factors are elements of a complex biological language,providing the basis for intercellular communication. They permit cellsto convey information between each other, mediate interaction betweencells and change gene expression. The effect of these multifunctionaland pluripotent factors is dependent on the presence or absence of otherpeptides.

FGF-8 is a member of the fibroblast growth factors (FGFs) which are afamily of heparin-binding, potent mitogens for both normal diploidfibroblasts and established cell lines, Gospodarowicz et al. (1984),Proc. Natl. Acad. Sci. USA 81:6963. The FGF family comprises acidic FGF(FGF-1), basic FGF (FGF-2), INT-2 (FGF-3), K-FGF/HST (FGF-4), FGF-5,FGF-6, KGF (FGF-7), AIGF (FGF-8) among others. All FGFs have twoconserved cysteine residues and share 30–50% sequence homology at theamino acid level. These factors are mitogenic for a wide variety ofnormal diploid mesoderm-derived and neural crest-derived cells,including granulosa cells, adrenal cortical cells, chondrocytes,myoblasts, corneal and vascular endothelial cells (bovine or human),vascular smooth muscle cells, lens, retina and prostatic epithelialcells, oligodendrocytes, astrocytes, chrondocytes, myoblasts andosteoblasts.

Fibroblast growth factors can also stimulate a large number of celltypes in a non-mitogenic manner. These activities include promotion ofcell migration into wound area (chemotaxis), initiation of new bloodvessel formulation (angiogenesis), modulation of nerve regeneration andsurvival (neurotrophism), modulation of endocrine functions, andstimulation or suppression of specific cellular protein expression,extracellular matrix production and cell survival. Baird & Bohlen,Handbook of Exp. Pharmacol. 95(1): 369–418, Springer, (1990). Theseproperties provide a basis for using fibroblast growth factors intherapeutic approaches to accelerate wound healing, nerve repair,collateral blood vessel formation, and the like. For example, fibroblastgrowth factors have been suggested to minimize myocardium damage inheart disease and surgery (U.S. Pat. No. 4,378,347).

FGF-8, also known as androgen-induced growth factor (AIGF), is a 215amino acid protein which shares 30–40% sequence homology with the othermembers of the FGF family. FGF-8 has been proposed to be underandrogenic regulation and induction in the mouse mammary carcinoma cellline SC3. Tanaka et al., Proc. Natl. Acad. Sci. USA 89: 8928–8932(1992); Sato et al., J. Steroid Biochem. Molec. Biol. 47: 91–98 (1993).As a result, FGF-8 may have a local role in the prostate, which is knownto be an androgen-responsive organ. FGF-8 can also be oncogenic, as itdisplays transforming activity when transfected into NIH-3T3fibroblasts. Kouhara et al., Oncogene 9 455–462 (1994). While FGF-8 hasbeen detected in heart, brain, lung, kidney, testis, prostate and ovary,expression was also detected in the absence of exogenous androgens.Schmitt et al., J. Steroid Biochem. Mol. Biol. 57 (3–4): 173–78 (1996).

FGF-8 shares the property with several other FGFs of being expressed ata variety of stages of murine embryogenesis, which supports the theorythat the various FGFs have multiple and perhaps coordinated roles indifferentiation and embryogenesis. Moreover, FGF-8 has also beenidentified as a protooncogene that cooperates with Wnt-1 in the processof mammary tumorigenesis (Shackleford et al., Proc. Natl. Acad. Sci. USA90, 740–744 (1993); Heikinheimo et al., Mech. Dev. 48: 129–138 (1994)).

In contrast to the other FGFs, FGF-8 exists as three protein isoforms,as a result of alternative splicing of the primary transcript. Tanaka etal., supra. Normal adult expression of FGF-8 is weak and confined togonadal tissue, however northern blot analysis has indicated that FGF-8mRNA is present from day 10 through day 12 or murine gestation, whichsuggests that FGF-8 is important to normal development. Heikinheimo etal., Mech Dev. 48(2): 129–38 (1994). Further in situ hybridizationassays between day 8 and 16 of gestation indicated initial expression inthe surface ectoderm of the first bronchial arches, the frontonasalprocess, the forebrain and the midbrain-hindbrain junction. At days10–12, FGF-8 was expressed in the surface ectoderm of the forelimb andhindlimb buds, the nasal its and nasopharynx, the infundibulum and inthe telencephalon, diencephalon and metencephalon. Expression continuesin the developing hindlimbs through day 13 of gestation, but isundetectable thereafter. The results suggest that FGF-8 has a uniquetemporal and spatial pattern in embryogenesis and suggests a role forthis growth factor in multiple regions of ectodermal differentiation inthe post-gastrulation embryo.

We herein describe the identification of novel poypeptides havinghomology to FGF-8, wherein those polypeptides are heein designatedPRO187 polypeptides.

SUMMARY OF THE INVENTION

Applicants have identified a cDNA clone that encodes a novelpolypeptide, designated in the present application as “PRO187”.

In one embodiment, the invention provides an isolated nucleic acidmolecule comprising DNA encoding a PRO187 polypeptide. In one aspect,the isolated nucleic acid comprises DNA encoding the PRO187 polypeptideof FIG. 2 (SEQ ID NO:2), or is complementary to such encoding nucleicacid sequence, and remains stably bound to it under at least moderate,and optionally, under high stringency conditions. In another aspect, theinvention provides a nucleic acid comprising the coding sequence of FIG.1 (SEQ ID NO:1) or its complement. In another aspect, the inventionprovides a nucleic acid of the full length protein of cloneDNA27864-1155, deposited with the American Type Culture Collection,Manassas, Va. (ATCC®) under accession number ATCC® 209375, alternativelythe coding sequence of clone DNA27864-1155, deposited under accessionnumber ATCC® 209375.

In yet another embodiment, the invention provides isolated PRO187polypeptide. In particular, the invention provides isolated nativesequence PRO187 polypeptide, which in one embodiment, includes an aminoacid sequence comprising residues 1 to 205 of FIG. 2 (SEQ ID NO:2).Alternatively, the invention provides a polypeptide encoded by thenucleic acid deposited under accession number ATCC® 209375.

Additional Embodiments

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody whichspecifically binds to any of the above or below described polypeptides.Optionally, the antibody is a monoclonal antibody, humanized antibody,antibody fragment or single-chain antibody.

In yet other embodiments, the invention provides oligonucleotide probesuseful for isolating genomic and cDNA nucleotide sequences, whereinthose probes may be derived from any of the above or below describednucleotide sequences.

In other embodiments, the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence that encodes a PROpolypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% sequence identity, preferably atleast about 81% sequence identity, more preferably at least about 82%sequence identity, yet more preferably at least about 83% sequenceidentity, yet more preferably at least about 84% sequence identity, yetmore preferably at least about 85% sequence identity, yet morepreferably at least about 86% sequence identity, yet more preferably atleast about 87% sequence identity, yet more preferably at least about88% sequence identity, yet more preferably at least about 89% sequenceidentity, yet more preferably at least about 90% sequence identity, yetmore preferably at least about 91% sequence identity, yet morepreferably at least about 92% sequence identity, yet more preferably atleast about 93% sequence identity, yet more preferably at least about94% sequence identity, yet more preferably at least about 95% sequenceidentity, yet more preferably at least about 96% sequence identity, yetmore preferably at least about 97% sequence identity, yet morepreferably at least about 98% sequence identity and yet more preferablyat least about 99% sequence identity to (a) a DNA molecule encoding aPRO polypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein or an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein, or (b) the complementof the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% sequence identity,preferably at least about 81% sequence identity, more preferably atleast about 82% sequence identity, yet more preferably at least about83% sequence identity, yet more preferably at least about 84% sequenceidentity, yet more preferably at least about 85% sequence identity, yetmore preferably at least about 86% sequence identity, yet morepreferably at least about 87% sequence identity, yet more preferably atleast about 88% sequence identity, yet more preferably at least about89% sequence identity, yet more preferably at least about 90% sequenceidentity, yet more preferably at least about 91% sequence identity, yetmore preferably at least about 92% sequence identity, yet morepreferably at least about 93% sequence identity, yet more preferably atleast about 94% sequence identity, yet more preferably at least about95% sequence identity, yet more preferably at least about 96% sequenceidentity, yet more preferably at least about 97% sequence identity, yetmore preferably at least about 98% sequence identity and yet morepreferably at least about 99% sequence identity to (a) a DNA moleculecomprising the coding sequence of a full-length PRO polypeptide cDNA asdisclosed herein, the coding sequence of a PRO polypeptide lacking thesignal peptide as disclosed herein or the coding sequence of anextracellular domain of a transmembrane PRO polypeptide, with or withoutthe signal peptide, as disclosed herein, or (b) the complement of theDNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%sequence identity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to (a) a DNAmolecule that encodes the same mature polypeptide encoded by any of thehuman protein cDNAs deposited with the ATCC® as disclosed herein, or (b)the complement of the DNA molecule of (a).

Another aspect the invention provides an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a PRO polypeptide which iseither transmembrane domain-deleted or transmembrane domain-inactivated,or is complementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PROpolypeptides are contemplated.

Another embodiment is directed to fragments of a PRO polypeptide codingsequence, or the complement thereof, that may find use as, for example,hybridization probes or for encoding fragments of a PRO polypeptide thatmay optionally encode a polypeptide comprising a binding site for ananti-PRO antibody. Such nucleic acid fragments are usually at leastabout 20 nucleotides in length, preferably at least about 30 nucleotidesin length, more preferably at least about 40 nucleotides in length, yetmore preferably at least about 50 nucleotides in length, yet morepreferably at least about 60 nucleotides in length, yet more preferablyat least about 70 nucleotides in length, yet more preferably at leastabout 80 nucleotides in length, yet more preferably at least about 90nucleotides in length, yet more preferably at least about 100nucleotides in length, yet more preferably at least about 110nucleotides in length, yet more preferably at least about 120nucleotides in length, yet more preferably at least about 130nucleotides in length, yet more preferably at least about 140nucleotides in length, yet more preferably at least about 150nucleotides in length, yet more preferably at least about 160nucleotides in length, yet more preferably at least about 170nucleotides in length, yet more preferably at least about 180nucleotides in length, yet more preferably at least about 190nucleotides in length, yet more preferably at least about 200nucleotides in length, yet more preferably at least about 250nucleotides in length, yet more preferably at least about 300nucleotides in length, yet more preferably at least about 350nucleotides in length, yet more preferably at least about 400nucleotides in length, yet more preferably at least about 450nucleotides in length, yet more preferably at least about 500nucleotides in length, yet more preferably at least about 600nucleotides in length, yet more preferably at least about 700nucleotides in length, yet more preferably at least about 800nucleotides in length, yet more preferably at least about 900nucleotides in length and yet more preferably at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PROpolypeptide-encoding nucleotide sequence may be determined in a routinemanner by aligning the PRO polypeptide-encoding nucleotide sequence withother known nucleotide sequences using any of a number of well knownsequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

In another embodiment, the invention provides isolated PRO polypeptideencoded by any of the isolated nucleic acid sequences hereinaboveidentified.

In a certain aspect, the invention concerns an isolated PRO polypeptide,comprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein or an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence having at least about 80% sequenceidentity, preferably at least about 81% sequence identity, morepreferably at least about 82% sequence identity, yet more preferably atleast about 83% sequence identity, yet more preferably at least about84% sequence identity, yet more preferably at least about 85% sequenceidentity, yet more preferably at least about 86% sequence identity, yetmore preferably at least about 87% sequence identity, yet morepreferably at least about 88% sequence identity, yet more preferably atleast about 89% sequence identity, yet more preferably at least about90% sequence identity, yet more preferably at least about 91% sequenceidentity, yet more preferably at least about 92% sequence identity, yetmore preferably at least about 93% sequence identity, yet morepreferably at least about 94% sequence identity, yet more preferably atleast about 95% sequence identity, yet more preferably at least about96% sequence identity, yet more preferably at least about 97% sequenceidentity, yet more preferably at least about 98% sequence identity andyet more preferably at least about 99% sequence identity to an aminoacid sequence encoded by any of the human protein cDNAs deposited withthe ATCC® as disclosed herein.

In a further aspect, the invention concerns an isolated PRO polypeptidecomprising an amino acid sequence scoring at least about 80% positives,preferably at least about 81% positives, more preferably at least about82% positives, yet more preferably at least about 83% positives, yetmore preferably at least about 84% positives, yet more preferably atleast about 85% positives, yet more preferably at least about 86%positives, yet more preferably at least about 87% positives, yet morepreferably at least about 88% positives, yet more preferably at leastabout 89% positives, yet more preferably at least about 90% positives,yet more preferably at least about 91% positives, yet more preferably atleast about 92% positives, yet more preferably at least about 93%positives, yet more preferably at least about 94% positives, yet morepreferably at least about 95% positives, yet more preferably at leastabout 96% positives, yet more preferably at least about 97% positives,yet more preferably at least about 98% positives and yet more preferablyat least about 99% positives when compared with the amino acid sequenceof a PRO polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein or an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein.

In a specific aspect, the invention provides an isolated PRO polypeptidewithout the N-terminal signal sequence and/or the initiating methionineand is encoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO polypeptide and recovering the PRO polypeptide from the cellculture.

Another aspect the invention provides an isolated PRO polypeptide whichis either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

In a further embodiment, the invention concerns a method of identifyingagonists or antagonists to a PRO polypeptide which comprise contactingthe PRO polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

In a still further embodiment, the invention concerns a composition ofmatter comprising a PRO polypeptide, or an agonist or antagonist of aPRO polypeptide as herein described, or an anti-PRO antibody, incombination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

Another embodiment of the present invention is directed to the use of aPRO polypeptide, or an agonist or antagonist thereof as hereinbeforedescribed, or an anti-PRO antibody, for the preparation of a medicamentuseful in the treatment of a condition which is responsive to the PROpolypeptide, an agonist or antagonist thereof or an anti-PRO antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO187 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA27864-1155”.

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods.

A “native sequence PRO polypeptide” comprises a polypeptide having thesame amino acid sequence as the corresponding PRO polypeptide derivedfrom nature. Such native sequence PRO polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native sequence PRO polypeptide” specifically encompassesnaturally-occurring truncated or secreted forms of the specific PROpolypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

The PRO polypeptide “extracellular domain” or “ECD” refers to a form ofthe PRO polypeptide which is essentially free of the transmembrane andcytoplasmic domains. Ordinarily, a PRO polypeptide ECD will have lessthan 1% of such transmembrane and/or cytoplasmic domains and preferably,will have less than 0.5% of such domains. It will be understood that anytransmembrane domains identified for the PRO polypeptides of the presentinvention are identified pursuant to criteria routinely employed in theart for identifying that type of hydrophobic domain. The exactboundaries of a transmembrane domain may vary but most likely by no morethan about 5 amino acids at either end of the domain as initiallyidentified herein. Optionally, therefore, an extracellular domain of aPRO polypeptide may contain from about 5 or fewer amino acids on eitherside of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are comtemplated by the present invention.

The approximate location of the “signal peptides” of the various PROpolypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1–6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683–4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

“PRO polypeptide variant” means an active PRO polypeptide as definedabove or below having at least about 80% amino acid sequence identitywith a full-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, preferably at least about 81% amino acid sequence identity,more preferably at least about 82% amino acid sequence identity, morepreferably at least about 83% amino acid sequence identity, morepreferably at least about 84% amino acid sequence identity, morepreferably at least about 85% amino acid sequence identity, morepreferably at least about 86% amino acid sequence identity, morepreferably at least about 87% amino acid sequence identity, morepreferably at least about 88% amino acid sequence identity, morepreferably at least about 89% amino acid sequence identity, morepreferably at least about 90% amino acid sequence identity, morepreferably at least about 91% amino acid sequence identity, morepreferably at least about 92% amino acid sequence identity, morepreferably at least about 93% amino acid sequence identity, morepreferably at least about 94% amino acid sequence identity, morepreferably at least about 95% amino acid sequence identity, morepreferably at least about 96% amino acid sequence identity, morepreferably at least about 97% amino acid sequence identity, morepreferably at least about 98% amino acid sequence identity and mostpreferably at least about 99% amino acid sequence identity with afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, often at least about 20 amino acids inlength, more often at least about 30 amino acids in length, more oftenat least about 40 amino acids in length, more often at least about 50amino acids in length, more often at least about 60 amino acids inlength, more often at least about 70 amino acids in length, more oftenat least about 80 amino acids in length, more often at least about 90amino acids in length, more often at least about 100 amino acids inlength, more often at least about 150 amino acids in length, more oftenat least about 200 amino acids in length, more often at least about 300amino acids in length, or more.

“Percent (%) amino acid sequence identity” with respect to the PROpolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460–480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

Percent amino acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

PRO variant polynucleotide” or “PRO variant nucleic acid sequence” meansa nucleic acid molecule which encodes an active PRO polypeptide asdefined below and which has at least about 80% nucleic acid sequenceidentity with a nucleotide acid sequence encoding a full-length nativesequence PRO polypeptide sequence as disclosed herein, a full-lengthnative sequence PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO polypeptide sequence as disclosed herein. Ordinarily,a PRO variant polynucleotide will have at least about 80% nucleic acidsequence identity, more preferably at least about 81% nucleic acidsequence identity, more preferably at least about 82% nucleic acidsequence identity, more preferably at least about 83% nucleic acidsequence identity, more preferably at least about 84% nucleic acidsequence identity, more preferably at least about 85% nucleic acidsequence identity, more preferably at least about 86% nucleic acidsequence identity, more preferably at least about 87% nucleic acidsequence identity, more preferably at least about 88% nucleic acidsequence identity, more preferably at least about 89% nucleic acidsequence identity, more preferably at least about 90% nucleic acidsequence identity, more preferably at least about 91% nucleic acidsequence identity, more preferably at least about 92% nucleic acidsequence identity, more preferably at least about 93% nucleic acidsequence identity, more preferably at least about 94% nucleic acidsequence identity, more preferably at least about 95% nucleic acidsequence identity, more preferably at least about 96% nucleic acidsequence identity, more preferably at least about 97% nucleic acidsequence identity, more preferably at least about 98% nucleic acidsequence identity and yet more preferably at least about 99% nucleicacid sequence identity with a nucleic acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, often at least about 60 nucleotides in length,more often at least about 90 nucleotides in length, more often at leastabout 120 nucleotides in length, more often at least about 150nucleotides in length, more often at least about 180 nucleotides inlength, more often at least about 210 nucleotides in length, more oftenat least about 240 nucleotides in length, more often at least about 270nucleotides in length, more often at least about 300 nucleotides inlength, more often at least about 450 nucleotides in length, more oftenat least about 600 nucleotides in length, more often at least about 900nucleotides in length, or more.

“Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460–480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

Percent nucleic acid sequence identity may also be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389–3402 (1997)). NCBI-BLAST2 uses several search parameters,wherein all of those search parameters are set to default valuesincluding, for example, unmask=yes, strand=all, expected occurrences=10,minimum low complexity length=15/5, multi-pass e-value=0.01, constantfor multi-pass=25, dropoff for final gapped alignment=25 and scoringmatrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons,the % nucleic acid sequence identity of a given nucleic acid sequence Cto, with, or against a given nucleic acid sequence D (which canalternatively be phrased as a given nucleic acid sequence C that has orcomprises a certain % nucleic acid sequence identity to, with, oragainst a given nucleic acid sequence D) is calculated as follows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program NCBI-BLAST2 in that program's alignment of Cand D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

In other embodiments, PRO variant polynucleotides are nucleic acidmolecules that encode an active PRO polypeptide and which are capable ofhybridizing, preferably under stringent hybridization and washconditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

The term “positives”, in the context of sequence comparison performed asdescribed above, includes residues in the sequences compared that arenot identical but have similar properties (e.g. as a result ofconservative substitutions, see Table 6 below). For purposes herein, the% value of positives is determined by dividing (a) the number of aminoacid residues scoring a positive value between the PRO polypeptide aminoacid sequence of interest having a sequence derived from the native PROpolypeptide sequence and the comparison amino acid sequence of interest(i.e., the amino acid sequence against which the PRO polypeptidesequence is being compared) as determined in the BLOSUM62 matrix ofWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest.

Unless specifically stated otherwise, the % value of positives iscalculated as described in the immediately preceding paragraph. However,in the context of the amino acid sequence identity comparisons performedas described for ALIGN-2 and NCBI-BLAST-2 above, includes amino acidresidues in the sequences compared that are not only identical, but alsothose that have similar properties. Amino acid residues that score apositive value to an amino acid residue of interest are those that areeither identical to the amino acid residue of interest or are apreferred substitution (as defined in Table 6 below) of the amino acidresidue of interest.

For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the %value of positives of a given amino acid sequence A to, with, or againsta given amino acid sequence B (which can alternatively be phrased as agiven amino acid sequence A that has or comprises a certain % positivesto, with, or against a given amino acid sequence B) is calculated asfollows:100 times the fraction X/Ywhere X is the number of amino acid residues scoring a positive value asdefined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2in that program's alignment of A and B, and where Y is the total numberof amino acid residues in B. It will be appreciated that where thelength of amino acid sequence A is not equal to the length of amino acidsequence B, the % positives of A to B will not equal the % positives ofB to A.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO monoclonal antibodies (includingagonist, antagonist, and neutralizing antibodies), anti-PRO antibodycompositions with polyepitopic specificity, single chain anti-PROantibodies, and fragments of anti-PRO antibodies (see below). The term“monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50M sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37–50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

“Active” or “activity” for the purposes herein refers to form(s) of aPRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats,rabbits, etc. Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10):1057–1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269–315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444–6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

By “solid phase” is meant a non-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

“PRO317-associated disorder” refers to a pathological condition ordisease wherein PRO317 is over- or under-expressed. Such disordersinclude diseases of the female genital tract or of the endometrium of amammal, including hyperplasia, endometritis, endometriosis, wherein thepatient is at risk for infertility due to endometrial factor,endometrioma, and endometrial cancer, especially those diseasesinvolving abnormal bleeding such as a gynecological disease. They alsoinclude diseases involving angiogenesis, wherein the angiogenesisresults in a pathological condition, such as cancer involving solidtumors (the therapy for the disorder would result in decreasedvascularization and a decline in growth and metastasis of a variety oftumors). Alternatively, the angiogenesis may be beneficial, such as forischemia, especially coronary ischemia. Hence, these disorders includethose found in patients whose hearts are functioning but who have ablocked blood supply due to atherosclerotic coronary artery disease, andthose with a functioning but underperfused heart, including patientswith coronary arterial disease who are not optimal candidates forangioplasty and coronary artery by-pass surgery. The disorders alsoinclude diseases involving the kidney or originating from the kidneytissue, such as polycystic kidney disease and chronic and acute renalfailure.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%II. Compositions and Methods of the Invention

A. Full-Length PRO Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO polypeptides. In particular, cDNAs encoding various PROpolypeptides have been identified and isolated, as disclosed in furtherdetail in the Examples below. It is noted that proteins produced inseparate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC®. The actual nucleotide sequences of thoseclones can readily be determined by the skilled artisan by sequencing ofthe deposited clone using routine methods in the art. The predictedamino acid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

-   -   Full-Length PRO187 Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO187. In particular, Applicants have identified and isolated cDNAencoding a PRO187 polypeptide, as disclosed in further detail in theExamples below. Using BLAST and FastA sequence alignment computerprograms, Applicants found that a full-length native sequence PRO187(shown in FIG. 15) has 74% amino acid sequence identity and BLAST scoreof 310 with various androgen-induced growth factors and FGF-8.Accordingly, it is presently believed that PRO187 polypeptide disclosedin the present application is a newly identified member of the FGF-8protein family and may possess identify activity or property typical ofthe FGF-8-like protein family.

B. PRO Polypeptide Variants

In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

Variations in the native full-length sequence PRO or in various domainsof the PRO described herein, can be made, for example, using any of thetechniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

PRO polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest areshown in Table 6 under the heading of preferred substitutions. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his;lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) aspasp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T)ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile;leu; met; phe; leu ala; norleucine

Substantial modifications in function or immunological identity of thePRO polypeptide are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

-   (1) hydrophobic: norleucine, met, ala, val, leu, ile;-   (2) neutral hydrophilic: cys, ser, thr;-   (3) acidic: asp, glu;-   (4) basic: asn, gln, his, lys, arg;-   (5) residues that influence chain orientation: gly, pro; and-   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081–1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

-   -   C. Modifications of PRO

Covalent modifications of PRO are included within the scope of thisinvention. One type of covalent modification includes reacting targetedamino acid residues of a PRO polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or the N- orC-terminal residues of the PRO. Derivatization with bifunctional agentsis useful, for instance, for crosslinking PRO to a water-insolublesupport matrix or surface for use in the method for purifying anti-PROantibodies, and vice-versa. Commonly used crosslinking agents include,e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79–86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259–306 (1981).

Removal of carbohydrate moieties present on the PRO polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO comprises linking the PROpolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, inthe manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192 or 4,179,337.

The PRO of the present invention may also be modified in a way to form achimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

In one embodiment, such a chimeric molecule comprises a fusion of thePRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159–2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610–3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547–553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204–1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192–194 (1992)]; an α-tubulin epitope peptide [Skinneret al. J. Biol. Chem., 266:15163–15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393–6397 (1990)].

In an alternative embodiment, the chimeric molecule may comprise afusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

-   -   D. Preparation of PRO

The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et. al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149–2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

-   -   -   1. Isolation of DNA Encoding PRO

DNA encoding PRO may be obtained from a cDNA library prepared fromtissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

Libraries can be screened with probes (such as antibodies to the PRO oroligonucleotides of at least about 20–80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

-   -   -   2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456–457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA) 76:3829 (1979). However, other methodsfor introducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185:527–537 (1990) and Mansour et al., Nature,336:348–352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC® 31,446); E. coliX1776 (ATCC® 31,537); E. coli strain W3110 (ATCC® 27,325) and K5 772(ATCC® 53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may, be modified toeffect a genetic mutation in the genes encoding proteins endogenous tothe host, with examples of such hosts including E. coli W3110 strain1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4,which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7(ATCC® 55,244), which has the complete genotype tonA ptr3phoA E15(argF-lac) 169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for PRO-encodingvectors. Saccharomyces cerevisiae is a commonly used lower eukaryotichost microorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology. 9:968–975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol. 737 [1983]), K.fragilis (ATCC® 12,424), K. bulgaricus (ATCC® 16,045), K. wickeramii(ATCC® 24,178), K. waltii (ATCC® 56,500), K. drosophilarum (ATCC®36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265–278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259–5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284–289 [1983]; Tilburn et al., Gene,26:205–221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470–1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475–479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO are derivedfrom multicellular organisms. Examples of invertebrate cells includeinsect cells such as Drosophila S2 and Spodoptera Sf9, as well as plantcells. Examples of useful mammalian host cell lines include Chinesehamster ovary (CHO) and COS cells. More specific examples include monkeykidney CV1 line transformed by SV40 (COS-7, ATCC® CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinesehamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Ac ad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod., 23:243–251 (1980)); human lung cells (W138, ATCC® CCL 75);human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC® CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

-   -   -   3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

The PRO may be produced recombinantly not only directly, but also as afusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC® No. 44076 orPEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21–25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg. 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO transcription from vectors in mammalian host cells is controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO by higher eukaryotes may beincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100–270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO in recombinant vertebrate cell culture aredescribed in Gething et al., Nature, 293:620–625 (1981); Mantei et al.,Nature, 281:40–46 (1979); EP 117,060; and EP 117,058.

-   -   -   4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201–5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

-   -   -   5. Purification of Polypeptide

Forms of PRO may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,SEPHADEX® G-75; PROTEIN A-SEPHAROSE™ (Pharmacia Biotech AB, Uppsala,Sweden) columns to remove contaminants such as IgG; and metal chelatingcolumns to bind epitope-tagged forms of the PRO. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular PRO produced.

-   -   E. Uses for PRO

Nucleotide sequences (or their complement) encoding PRO have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

The full-length native sequence PRO gene, or portions thereof, may beused as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO nucleic acids include antisense orsense oligonucleotides comprising a singe-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target PRO mRNA (sense) or PRODNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO DNA. Such a fragment generally comprises at least about 14nucleotides, preferably from about 14 to 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide, based upon a cDNAsequence encoding a given protein is described in, for example, Steinand Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO proteins.Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense RNA or DNA molecules are generally at least about 5 bases inlength, about 10 bases in length, about 15 bases in length, about 20bases in length, about 25 bases in length, about 30 bases in length,about 35 bases in length, about 40 bases in length, about 45 bases inlength, about 50 bases in length, about 55 bases in length, about 60bases in length, about 65 bases in length, about 70 bases in length,about 75 bases in length, about 80 bases in length, about 85 bases inlength, about 90 bases in length, about 95 bases in length, about 100bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO coding sequences.

Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

Nucleic acids which encode PRO or its modified forms can also be used togenerate either transgenic animals or “knock out” animals which, inturn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PRO can be used to construct aPRO “knock out” animal which has a defective or altered gene encodingPRO as a result of homologous recombination between the endogenous geneencoding PRO and altered genomic DNA encoding PRO introduced into anembryonic stem cell of the animal. For example, cDNA encoding PRO can beused to clone genomic DNA encoding PRO in accordance with establishedtechniques. A portion of the genomic DNA encoding PRO can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113–152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the PROpolypeptide.

Nucleic acid encoding the PRO polypeptides may also be used in genetherapy. In gene therapy applications, genes are introduced into cellsin order to achieve in vivo synthesis of a therapeutically effectivegenetic product, for example for replacement of a defective gene. “Genetherapy” includes both conventional gene therapy where a lasting effectis achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143–4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205–210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429–4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410–3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808–813 (1992).

The PRO polypeptides described herein may also be employed as molecularweight markers for protein electrophoresis purposes and the isolatednucleic acid sequences may be used for recombinantly expressing thosemarkers.

The nucleic acid molecules encoding the PRO polypeptides or fragmentsthereof described herein are useful for chromosome identification. Inthis regard, there exists an ongoing need to identify new chromosomemarkers, since relatively few chromosome marking reagents, based uponactual sequence data are presently available. Each PRO nucleic acidmolecule of the present invention can be used as a chromosome marker.

The PRO polypeptides and nucleic acid molecules of the present inventionmay also be used for tissue typing, wherein the PRO polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another. PRO nucleic acid molecules will find use forgenerating probes for PCR, Northern analysis, Southern analysis andWestern analysis.

The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrateand other organic acids; antioxidants including ascorbic acid; lowmolecular weight (less than about 10 residues) polypeptides; proteins,such as serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose, or dextrins; chelating agentssuch as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formingcounterions such as sodium; and/or nonionic surfactants such as TWEEN™,PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42–96.

When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

Where sustained-release administration of a PRO polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795–799 (1996); Yasuda, Biomed. Ther., 27:1221–1223 (1993); Horaet al., Bio/Technology 8:755–758 (1990); Cleland, “Design and Productionof Single Immunization Vaccines Using Polylactide PolyglycolideMicrosphere Systems,” in Vaccine Design: The Subunit and AdjuvantApproach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp.439–462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No.5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1–41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO polypeptide (agonists) or prevent the effect ofthe PRO polypeptide (antagonists). Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO polypeptides encoded by the genes identified herein, orotherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the PRO polypeptide encoded by the gene identified herein orthe drug candidate is immobilized on a solid phase, e.g., on amicrotiter plate, by covalent or non-covalent attachments. Non-covalentattachment generally is accomplished by coating the solid surface with asolution of the PRO polypeptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for the PROpolypeptide to be immobilized can be used to anchor it to a solidsurface. The assay is performed by adding the non-immobilized component,which may be labeled by a detectable label, to the immobilizedcomponent, e.g., the coated surface containing the anchored component.When the reaction is complete, the non-reacted components are removed,e.g., by washing, and complexes anchored on the solid surface aredetected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London) 340:245–246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578–9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789–5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a PROpolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the PRO polypeptide may be added to a cellalong with the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeled PROpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Crosslinked material isresolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

Another potential PRO polypeptide antagonist is an antisense RNA or DNAconstruct prepared using antisense technology, where, e.g., an antisenseRNA or DNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO polypeptide, thereby blocking the normalbiological activity of the PRO polypeptide. Examples of small moleculesinclude, but are not limited to, small peptides or peptide-likemolecules, preferably soluble peptides, and synthetic non-peptidylorganic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology 4:469–471 (1994), and PCT publication No. WO 97/33551 (publishedSep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

With regard to the PRO211 and PRO217 polypeptide, therapeuticindications include disorders associated with the preservation andmaintenance of gastrointestinal mucosa and the repair of acute andchronic mucosal lesions (e.g., enterocolitis, Zollinger-Ellisonsyndrome, gastrointestinal ulceration and congenital microvillusatrophy), skin diseases associated with abnormal keratinocytedifferentiation (e.g., psoriasis, epithelial cancers such as lungsquamous cell carcinoma, epidermoid carcinoma of the vulva and gliomas.

Since the PRO232 polypeptide and nucleic acid encoding it possesssequence homology to a cell surface stem cell antigen and its encodingnucleic acid, probes based upon the PRO232 nucleotide sequence may beemployed to identify other novel stem cell surface antigen proteins.Soluble forms of the PRO232 polypeptide may be employed as antagonistsof membrane bound PRO232 activity both in vitro and in vivo. PRO232polypeptides may be employed in screening assays designed to identifyagonists or antagonists of the native PRO232 polypeptide, wherein suchassays may take the form of any conventional cell-type or biochemicalbinding assay. Moreover, the PRO232 polypeptide may serve as a molecularmarker for the tissues in which the polypeptide is specificallyexpressed.

With regard to the PRO187 polypeptides disclosed herein, FGF-8 has beenimplicated in cellular differentiation and embryogenesis, including thepatterning which appears during limb formation. FGF-8 and the PRO187molecules of the invention therefore are likely to have potent effectson cell growth and development. Diseases which relate to cellular growthand differentiation are therefore suitable targets for therapeuticsbased on functionality similar to FGF-8. For example, diseases relatedto growth or survival of nerve cells including Parkinson's disease,Alzheimer's disease, ALS, neuropathies. Additionally, disease related touncontrolled cell growth, e.g., cancer, would also be expectedtherapeutic targets.

With regard to the PRO265 polypeptides disclosed herein, other methodsfor use with PRO265 are described in U.S. Pat. No. 5,654,270 toRuoslahti et al. In particular, PRO265 can be used in comparison withthe fibromodulin disclosed therein to compare its effects on reducingdermal scarring and other properties of the fibromodulin describedtherein including where it is located and with what it binds and doesnot.

The PRO219 polypeptides of the present invention which play a regulatoryrole in the blood coagulation cascade may be employed in vivo fortherapeutic purposes as well as for in vitro purposes. Those of ordinaryskill in the art will well know how to employ PRO219 polypeptides forsuch uses.

The PRO246 polypeptides of the present invention which serve as cellsurface receptors for one or more viruses will find other uses. Forexample, extracellular domains derived from these PRO246 polypeptidesmay be employed therapeutically in vivo for lessening the effects ofviral infection. Those PRO246 polypeptides which serves as tumorspecific antigens may be exploited as therapeutic targets for anti-tumordrugs, and the like. Those of ordinary skill in the art will well knowhow to employ PRO246 polypeptides for such uses.

Assays in which connective growth factor and other growth factors areusually used should be performed with PRO261. An assay to determinewhether TGF beta induces PRO261, indicating a role in cancer isperformed as known in the art. Wound repair and tissue growth assays arealso performed with PRO261. The results are applied accordingly.

PRO228 polypeptides should be used in assays in which EMR1, CD97 andlatrophilin would be used in to determine their relative activities. Theresults can be applied accordingly. For example, a competitive bindingassay with PRO228 and CD97 can be performed with the ligand for CD97,CD55.

Native PRO533 is a 216 amino acid polypeptide of which residues 1–22 arethe signal sequence. Residues 3 to 216 have a Blast score of 509,corresponding to 53% homology to fibroblast growth factor. At thenucleotide level, DNA47412, the EST from which PCR oligos were generatedto isolate the full length DNA49435-1219, has been observed to map to11p15. Sequence homology to the 11p15locus would indicate that PRO533may have utility in the treatment of Usher Syndrome or Atrophia areata.

As mentioned previously, fibroblast growth factors can act upon cells inboth a mitogenic and non-mitogenic manner. These factors are mitogenicfor a wide variety of normal diploid mesoderm-derived and neuralcrest-derived cells, inducing granulosa cells, adrenal cortical cells,chrondrocytes, myoblasts, corneal and vascular endothelial cells (bovineor human), vascular smooth muscle cells, lens, retina and prostaticepithelial cells, oligodendrocytes, astrocytes, chrondocytes, myoblastsand osteoblasts.

Non-mitogenic actions of fibroblast growth factors include promotion ofcell migration into a wound area (chemotaxis), initiation of new bloodvessel formulation (angiogenesis), modulation of nerve regeneration andsurvival (neurotrophism), modulation of endocrine functions, andstimulation or suppression of specific cellular protein expression,extracellular matrix production and cell survival. Baird, A. & Bohlen,P., Handbook of Exp. Phrmacol. 95(1):369–418 (1990). These propertiesprovide a basis for using fibroblast growth factors in therapeuticapproaches to accelerate wound healing, nerve repair, collateral bloodvessel formation, and the like. For example, fibroblast growth factors,have been suggested to minimize myocardium damage in heart disease andsurgery (U.S. Pat. No. 4,378,437).

Since the PRO245 polypeptide and nucleic acid encoding it possesssequence homology to a transmembrane protein tyrosine kinase protein andits encoding nucleic acid, probes based upon the PRO245 nucleotidesequence may be employed to identify other novel transmembrane tyrosinekinase proteins. Soluble forms of the PRO245 polypeptide may be employedas antagonists of membrane bound PRO245 activity both in vitro and invivo. PRO245 polypeptides may be employed in screening assays designedto identify agonists or antagonists of the native PRO245 polypeptide,wherein such assays may take the form of any conventional cell-type orbiochemical binding assay. Moreover, the PRO245 polypeptide may serve asa molecular marker for the tissues in which the polypeptide isspecifically expressed.

PRO220, PRO221 and PRO227 all have leucine rich repeats. Additionally,PRO220 and PRO221 have homology to SLIT and leucine rich repeat protein.Therefore, these proteins are useful in assays described in theliterature, supra, wherein the SLIT and leucine rich repeat protein areused. Regarding the SLIT protein, PRO227 can be used in an assay todetermine the affect of PRO227 on neurodegenerative disease.Additionally, PRO227 has homology to human glycoprotein V. In the caseof PRO227, this polypeptide is used in an assay to determine its affecton bleeding, clotting, tissue repair and scarring.

The PRO266 polypeptide can be used in assays to determine if it has arole in neurodegenerative diseases or their reversal.

PRO269 polypeptides and portions thereof which effect the activity ofthrombin may also be useful for in vivo therapeutic purposes, as well asfor various in vitro applications. In addition, PRO269 polypeptides andportions thereof may have therapeutic use as an antithrombotic agentwith reduced risk for hemorrhage as compared with heparin. Peptideshaving homology to thrombomodulin are particularly desirable.

PRO287 polypeptides and portions thereof which effect the activity ofbone morphogenic protein “BMP1”/procollagen C-proteinase (PCP) may alsobe useful for in vivo therapeutic purposes, as well as for various invitro applications. In addition, PRO287 polypeptides and portionsthereof may have therapeutic applications in wound healing and tissuerepair. Peptides having homology to procollagen C-proteinase enhancerprotein and its precursor may also be used to induce bone and/orcartilage formation and are therefore of particular interest to thescientific and medical communities.

Therapeutic indications for PRO214 polypeptides include disordersassociated with the preservation and maintenance of gastrointestinalmucosa and the repair of acute and chronic mucosal lesions (e.g.,enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcerationand congenital microvillus atrophy), skin diseases associated withabnormal keratinocyte differentiation (e.g., psoriasis, epithelialcancers such as lung squamous cell carcinoma, epidermoid carcinoma ofthe vulva and gliomas.

Studies on the generation and analysis of mice deficient in members ofthe TGF-superfamily are reported in Matzuk, Trends in Endocrinol. andMetabol., 6: 120–127 (1995).

The PRO317 polypeptide, as well as PRO317-specific antibodies,inhibitors, agonists, receptors, or their analogs, herein are useful intreating PRO317-associated disorders. Hence, for example, they may beemployed in modulating endometrial bleeding angiogenesis, and may alsohave an effect on kidney tissue. Endometrial bleeding can occur ingynecological diseases such as endometrial cancer as abnormal bleeding.Thus, the compositions herein may find use in diagnosing and treatingabnormal bleeding conditions in the endometrium, as by reducing oreliminating the need for a hysterectomy. The molecules herein may alsofind use in angiogenesis applications such as anti-tumor indications forwhich the antibody against vascular endothelial growth factor is used,or, conversely, ischemic indications for which vascular endothelialgrowth factor is employed.

Bioactive compositions comprising PRO317 or agonists or antagoniststhereof may be administered in a suitable therapeutic dose determined byany of several methodologies including clinical studies on mammalianspecies to determine maximal tolerable dose and on normal human subjectsto determine safe dose. Additionally, the bioactive agent may becomplexed with a variety of well established compounds or compositionswhich enhance stability or pharmacological properties such as half-life.It is contemplated that the therapeutic, bioactive composition may bedelivered by intravenous infusion into the bloodstream or any othereffective means which could be used for treating problems of the kidney,uterus, endometrium, blood vessels, or related tissue, e.g., in theheart or genital tract.

Dosages and administration of PRO317, PRO317 agonist, or PRO317antagonist in a pharmaceutical composition may be determined by one ofordinary skill in the art of clinical pharmacology or pharmacokinetics.See, for example, Mordenti and Rescigno, Pharmaceutical Research,9:17–25 (1992); Morenti et al., Pharmaceutical Research, 8:1351–1359(1991); and Mordenti and Chappell, “The use of interspecies scaling intoxicokinetics” in Toxicokinetics and New Drug Development, Yacobi etal. (eds) (Pergamon Press: NY, 1989), pp. 42–96. An effective amount ofPRO317, PRO317 agonist, or PRO317 antagonist to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of themammal. Accordingly, it will be necessary for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 10 ng/kg to up to 100 mg/kg of the mammal's body weight or moreper day, preferably about 1 μg/kg/day to 10 mg/kg/day. Typically, theclinician will administer PRO317, PRO317 agonist, or PRO317 antagonist,until a dosage is reached that achieves the desired effect for treatmentof the above mentioned disorders.

PRO317 or an PRO317 agonist or PRO317 antagonist may be administeredalone or in combination with another to achieve the desiredpharmacological effect. PRO317 itself, or agonists or antagonists ofPRO317 can provide different effects when administered therapeutically.Such compounds for treatment will be formulated in a nontoxic, inert,pharmaceutically acceptable aqueous carrier medium preferably at a pH ofabout 5 to 8, more preferably 6 to 8, although the pH may vary accordingto the characteristics of the PRO317, agonist, or antagonist beingformulated and the condition to be treated. Characteristics of thetreatment compounds include solubility of the molecule, half-life, andantigenicity/immunogenicity; these and other characteristics may aid indefining an effective carrier.

PRO317 or PRO317 agonists or PRO317 antagonists may be delivered byknown routes of administration including but not limited to topicalcreams and gels; transmucosal spray and aerosol, transdermal patch andbandage; injectable; intravenous, and lavage formulations; and orallyadministered liquids and pills, particularly formulated to resiststomach acid and enzymes. The particular formulation, exact dosage, androute of administration will be determined by the attending physicianand will vary according to each specific situation.

Such determinations of administration are made by considering multiplevariables such as the condition to be treated, the type of mammal to betreated, the compound to be administered, and the pharmacokineticprofile of the particular treatment compound. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time of administration, drugcombination, reaction sensitivities, and tolerance/response to therapy.Long-acting treatment compound formulations (such as liposomallyencapsulated PRO317 or PEGylated PRO317 or PRO317 polymericmicrospheres, such as polylactic acid-based microspheres) might beadministered every 3 to 4 days, every week, or once every two weeksdepending on half-life and clearance rate of the particular treatmentcompound.

Normal dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg ofmammal body weight or more per day, preferably about 1 μg/kg/day to 10mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting the uterus, for example, may necessitatedelivery in a manner different from that to another organ or tissue,such as cardiac tissue.

Where sustained-release administration of PRO317 is desired in aformulation with release characteristics suitable for the treatment ofany disease or disorder requiring administration of PRO317,microencapsulation of PRO317 is contemplated. Microencapsulation ofrecombinant proteins for sustained release has been successfullyperformed with human growth hormone (rhGH), interferon-(rhIFN-),interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2: 795–799(1996); Yasuda Biomed. Ther., 27: 1221–1223 (1993); Hora et al.,Bio/Technology, 8: 755–758 (1990); Cleland, “Design and Production ofSingle Immunization Vaccines Using Polylactide Polyglycolide MicrosphereSystems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powelland Newman, eds, (Plenum Press: New York, 1995), pp. 439–462; WO97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No. 5,654,010.

It is contemplated that conditions or diseases of the uterus,endometrial tissue, or other genital tissues or cardiac tissues mayprecipitate damage that is treatable with PRO317 or PRO317 agonist wherePRO317 expression is reduced in the diseased state; or with antibodiesto PRO317 or other PRO317 antagonists where the expression of PRO317 isincreased in the diseased state. These conditions or diseases may bespecifically diagnosed by the probing tests discussed above forphysiologic and pathologic problems which affect the function of theorgan.

The PRO317, PRO317 agonist, or PRO317 antagonist may be administered toa mammal with another biologically active agent, either separately or inthe same formulation to treat a common indication for which they areappropriate. For example, it is contemplated that PRO317 can beadministered together with EBAF-1 for those indications on which theydemonstrate the same qualitative biological effects. Alternatively,where they have opposite effects, EBAF-1 may be administered togetherwith an antagonist to PRO317, such as an anti-PRO317 antibody. Further,PRO317 may be administered together with VEGF for coronary ischemiawhere such indication is warranted, or with an anti-VEGF for cancer aswarranted, or, conversely, an antagonist to PRO317 may be administeredwith VEGF for coronary ischemia or with anti-VEGF to treat cancer aswarranted. These administrations would be in effective amounts fortreating such disorders.

Native PRO301 has a Blast score of 246 and 30% homology at residues 24to 282 of FIG. 44 with A33_HUMAN, an A33 antigen precursor. A33 antigenprecursor, as explained in the Background is a tumor-specific antigen,and as such, is a recognized marker and therapeutic target for thediagnosis and treatment of colon cancer. The expression oftumor-specific antigens is often associated with the progression ofneoplastic tissue disorders. Native PRO301 and A33_HUMAN also show aBlast score of 245 and 30% homology at residues 21 to 282 of FIG. 44with A33_HUMAN, the variation dependent upon how spaces are insertedinto the compared sequences. Native PRO301 also has a Blast score of 165and 29% homology at residues 60 to 255 of FIG. 44 with HS46 KDA_(—)1, ahuman coxsackie and adenovirus receptor protein, also known as cellsurface protein HCAR. This region of PRO301 also shows a similar Blastscore and homology with HSU90716_(—)1. Expression of such proteins isusually associated with viral infection and therapeutics for theprevention of such infection may be accordingly conceived. As mentionedin the Background, the expression of viral receptors is often associatedwith neoplastic tumors.

Therapeutic uses for the PRO234 polypeptides of the invention includestreatments associated with leukocyte homing or the interaction betweenleukocytes and the endothelium during an inflammatory response. Examplesinclude asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.

Since the PRO231 polypeptide and nucleic acid encoding it possesssequence homology to a putative acid phosphatase and its encodingnucleic acid, probes based upon the PRO231 nucleotide sequence may beemployed to identify other novel phosphatase proteins. Soluble forms ofthe PRO231 polypeptide may be employed as antagonists of membrane boundPRO231 activity both in vitro and in vivo. PRO231 polypeptides may beemployed in screening assays designed to identify agonists orantagonists of the native PRO231 polypeptide, wherein such assays maytake the form of any conventional cell-type or biochemical bindingassay. Moreover, the PRO231 polypeptide may serve as a molecular markerfor the tissues in which the polypeptide is specifically expressed.

PRO229 polypeptides can be fused with peptides of interest to determinewhether the fusion peptide has an increased half-life over the peptideof interest. The PRO229 polypeptides can be used accordingly to increasethe half-life of polypeptides of interest. Portions of PRO229 whichcause the increase in half-life are an embodiment of the inventionherein.

PRO238 can be used in assays which measure its ability to reducesubstrates, including oxygen and Aceyl-CoA, and particularly, measurePRO238's ability to produce oxygen free radicals. This is done by usingassays which have been previously described. PRO238 can further be usedto assay for candidates which block, reduce or reverse its reducingabilities. This is done by performing side by side assays wherecandidates are added in one assay having PRO238 and a substrate toreduce, and not added in another assay, being the same but for the lackof the presence of the candidate.

PRO233 polypeptides and portions thereof which have homology toreductase may also be useful for in vivo therapeutic purposes, as wellas for various other applications. The identification of novel reductaseproteins and related molecules may be relevant to a number of humandisorders such as inflammatory disease, organ failure, atherosclerosis,cardiac injury, infertility, birth defects, premature aging, AIDS,cancer, diabetic complications and mutations in general. Given thatoxygen free radicals and antioxidants appear to play important roles ina number of disease processes, the identification of new reductaseproteins and reductase-like molecules is of special importance in thatsuch proteins may serve as potential therapeutics for a variety ofdifferent human disorders. Such polypeptides may also play importantroles in biotechnological and medical research, as well as variousindustrial applications. As a result, there is particular scientific andmedical interest in new molecules, such as PRO233.

The PRO223 polypeptides of the present invention which exhibit serinecarboxypeptidease activity may be employed in vivo for therapeuticpurposes as well as for in vitro purposes. Those of ordinary skill inthe art will well know how to employ PRO223 polypeptides for such uses.

PRO235 polypeptides and portions thereof which may be involved in celladhesion are also useful for in vivo therapeutic purposes, as well asfor various in vitro applications. In addition, PRO235 polypeptides andportions thereof may have therapeutic applications in disease stateswhich involve cell adhesion. Given the physiological importance of celladhesion mechanisms in vivo, efforts are currently being under taken toidentify new, native proteins which are involved in cell adhesion.Therefore, peptides having homology to plexin are of particular interestto the scientific and medical communities.

Because the PRO236 and PRO262 polypeptides disclosed herein arehomologous to various known β-galactosidase proteins, the PRO236 andPRO262 polypeptides disclosed herein will find use in conjugates ofmonoclonal antibodies and the polypeptide for specific killing of tumorcells by generation of active drug from a galactosylated prodrug (e.g.,the generation of 5-fluorouridine from the prodrugβ-D-galactosyl-5-fluorouridine). The PRO236 and PRO262 polypeptidesdisclosed herein may also find various uses both in vivo and in vitro,wherein those uses will be similar or identical to uses for whichβ-galactosidase proteins are now employed. Those of ordinary skill inthe art will well know how to employ PRO236 and PRO262 polypeptides forsuch uses.

PRO239 polypeptides and portions thereof which have homology to densinmay also be useful for in vivo therapeutic purposes, as well as forvarious in vitro applications. In addition, PRO239 polypeptides andportions thereof may have therapeutic applications in disease stateswhich involve synaptic mechanisms, regeneration or cell adhesion. Giventhe physiological importance of synaptic processes, regeneration andcell adhesion mechanisms in vivo, efforts are currently being undertaken to identify new, native proteins which are involved in synapticmachinery and cell adhesion. Therefore, peptides having homology todensin are of particular interest to the scientific and medicalcommunities.

The PRO260 polypeptides described herein can be used in assays todetermine their relation to fucosidase. In particular, the PRO260polypeptides can be used in assays in determining their ability toremove fucose or other sugar residues from proteoglycans. The PRO260polypeptides can be assayed to determine if they have any functional orlocational similarities as fucosidase. The PRO260 polypeptides can thenbe used to regulate the systems in which they are integral.

PRO263 can be used in assays wherein CD44 antigen is generally used todetermine PRO263 activity relative to that of CD44. The results can beused accordingly.

PRO270 polypeptides and portions thereof which effectreduction-oxidation (redox) state may also be useful for in vivotherapeutic purposes, as well as for various in vitro applications. Morespecifically, PRO270 polypeptides may affect the expression of a largevariety of genes thought to be involved in the pathogenesis of AIDS,cancer, atherosclerosis, diabetic complications and in pathologicalconditions involving oxidative stress such as stroke and inflammation.In addition, PRO270 polypeptides and portions thereof may affect theexpression of a genes which have a role in apoptosis. Therefore,peptides having homology to thioredoxin are particularly desirable tothe scientific and medical communities.

PRO272 polypeptides and portions thereof which possess the ability tobind calcium may also have numerous in vivo therapeutic uses, as well asvarious in vitro applications. Therefore, peptides having homology toreticulocalbin are particularly desirable. Those with ordinary skill inthe art will know how to employ PRO272 polypeptides and portions thereoffor such purposes.

PRO294 polypeptides and portions thereof which have homology to collagenmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel collagens andcollage-like molecules may have relevance to a number of humandisorders. Thus, the identification of new collagens and collage-likemolecules is of special importance in that such proteins may serve aspotential therapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as various industrial applications. Given thelarge number of uses for collagen, there is substantial interest inpolypeptides with homology to the collagen molecule.

PRO295 polypeptides and portions thereof which have homology to integrinmay also be useful for in vivo therapeutic purposes, as well as forvarious other applications. The identification of novel integrins andintegrin-like molecules may have relevance to a number of humandisorders such as modulating the binding or activity of cells of theimmune system. Thus, the identification of new integrins andintegrin-like molecules is of special importance in that such proteinsmay serve as potential therapeutics for a variety of different humandisorders. Such polypeptides may also play important roles inbiotechnological and medical research as well as various industrialapplications. As a result, there is particular scientific and medicalinterest in new molecules, such as PRO295.

As the PRO293 polypeptide is clearly a leucine rich repeat polypeptidehomologue, the peptide can be used in all applications that the knownNLRR-1 and NLRR-2 polypeptides are used. The activity can be comparedbetween these peptides and thus applied accordingly.

The PRO247 polypeptides described herein can be used in assays in whichdensin is used to determine the activity of PRO247 relative to densin orthese other proteins. The results can be used accordingly in diagnosticsand/or therapeutic applications with PRO247.

PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the presentinvention which possess protease activity may be employed both in vivofor therapeutic purposes and in vitro. Those of ordinary skill in theart will well know how to employ the PRO302, PRO303, PRO304, PRO307 andPRO343 polypeptides of the present invention for such purposes.

PRO328 polypeptides and portions thereof which have homology to GLIP andCRISP may also be useful for in vivo therapeutic purposes, as well asfor various other applications. The identification of novel GLIP andCRISP-like molecules may have relevance to a number of human disorderswhich involve transcriptional regulation or are over expressed in humantumors. Thus, the identification of new GLIP and CRISP-like molecules isof special importance in that such proteins may serve as potentialtherapeutics for a variety of different human disorders. Suchpolypeptides may also play important roles in biotechnological andmedical research as well as in various industrial applications. As aresult, there is particular scientific and medical interest in newmolecules, such as PRO328.

Uses for PRO335, PRO331 or PRO326 including uses in competitive assayswith LIG-1, ALS and decorin to determine their relative activities. Theresults can be used accordingly. PRO335, PRO331 or PRO326 can also beused in assays where LIG-1 would be used to determine if the sameeffects are incurred.

PRO332 contains GAG repeat (GKEK) at amino acid positions 625–628 inFIG. 108. Slippage in such repeats can be associated with human disease.Accordingly, PRO332 can use useful for the treatment of such diseaseconditions by gene therapy, i.e. by introduction of a gene containingthe correct GKEK sequence motif.

Other uses of PRO334 include use in assays in which fibrillin or fibulinwould be used to determine the relative activity of PRO334 to fibrillinor fibulin. In particular, PRO334 can be used in assays which requirethe mechanisms imparted by epidermal growth factor repeats.

Native PRO346 has a Blast score of 230, corresponding to 27% homologybetween amino acid residues 21 to 343 with residues 35 to 1040CGM6_HUMAN, a carcinoembryonic antigen cgm6 precursor. This homologyregion includes nearly all but 2 N-terminal extracellular domainresidues, including an immunoglobulin superfamily homology at residues148 to 339 of PRO346 in addition to several transmembrane residues(340–343). Carcinoembryonic antigen precursor, as explained in theBackground is a tumor-specific antigen, and as such, is a recognizedmarker and therapeutic target for the diagnosis and treatment of coloncancer. The expression of tumor-specific antigens is often associatedwith the progression of neoplastic tissue disorders. Native PRO346 andP_WO6874, a human carcinoembryonic antigen CEA-d have a Blast score of224 and homology of 28% between residues 2 to 343 and 67 to 342,respectively. This homology includes the entire extracellular domainresidues of native PRO346, minus the initiator methionine (residues 2 to18) as well as several transmembrane residues (340–343).

PRO268 polypeptides which have protein disulfide isomerase activity willbe useful for many applications where protein disulfide isomeraseactivity is desirable including, for example, for use in promotingproper disulfide bond formation in recombinantly produced proteins so asto increase the yield of correctly folded protein. Those of ordinaryskill in the art will readily know how to employ such PRO268polypeptides for such purposes.

PRO330 polypeptides of the present invention which possess biologicalactivity related to that of the prolyl 4-hydroxylase alpha subunitprotein may be employed both in vivo for therapeutic purposes and invitro. Those of ordinary skill in the art will well know how to employthe PRO330 polypeptides of the present invention for such purposes.

Uses of the herein disclosed molecules may also be based upon thepositive functional assay hits disclosed and described below.

-   -   F. Anti-PRO Antibodies

The present invention further provides anti-PRO antibodies. Exemplaryantibodies include polyclonal, monoclonal, humanized, bispecific, andheteroconjugate antibodies.

-   -   1. Polyclonal Antibodies

The anti-PRO antibodies may comprise polyclonal antibodies. Methods ofpreparing polyclonal antibodies are known to the skilled artisan.Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Theimmunizing agent may include the PRO polypeptide or a fusion proteinthereof. It may be useful to conjugate the immunizing agent to a proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants which may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). The immunization protocol may beselected by one skilled in the art without undue experimentation.

-   -   2. Monoclonal Antibodies

The anti-PRO antibodies may, alternatively, be monoclonal antibodies.Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include the PRO polypeptide or afusion protein thereof. Generally, either peripheral blood lymphocytes(“PBLs”) are used if cells of human origin are desired, or spleen cellsor lymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell [Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59–103]. Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,bovine and human origin. Usually, rat or mouse myeloma cell lines areemployed. The hybridoma cells may be cultured in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, immortalized cells. For example, ifthe parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection (ATCC®), Manassas, Va. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51–63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against PRO.Preferably, the binding specificity of monoclonal antibodies produced bythe hybridoma cells is determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollard,Anal. Biochem. 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, PROTEINA-SEPHAROSE™, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences [U.S. Pat.No. 4,816,567; Morrison et al., supra] or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

-   -   3. Human and Humanized Antibodies

The anti-PRO antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherint he recipient antibody nor int he imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522–525 (1986); Riechmann etal., Nature, 332:323–329 (1988); and Presta, Curr. Op. Struct. Biol.2:593–596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522–525 (1986); Riechmann et al., Nature,332:323–327 (1988); Verhoeyen et al., Science, 239:1534–1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86–95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779–783(1992); Lonberg et al., Nature 368 856–859 (1994); Morrison, Nature 368,812–13 (1994); Fishwild et al., Nature Biotechnology 14, 845–51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65–93 (1995).

-   -   4. Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forthe PRO, the other one is for any other antigen, and preferably for acell-surface protein or receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537–539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655–3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Fab′ fragments may be directly recovered from E. coli and chemicallycoupled to form bispecific antibodies. Shalaby et al., J. Exp. Med.175:217–225 (1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

Various technique for making and isolating bispecific antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bispecific antibodies have been produced using leucine zippers.Kostelny et al., J. Immunol. 148(5):1547–1553 (1992). The leucine zipperpeptides from the Fos and Jun proteins were linked to the Fab′ portionsof two different antibodies by gene fusion. The antibody homodimers werereduced at the hinge region to form monomers and then re-oxidized toform the antibody heterodimers. This method can also be utilized for theproduction of antibody homodimers. The “diabody” technology described byHollinger et al., Proc. Natl. Acad. Sci. USA 90:6444–6448 (1993) hasprovided an alternative mechanism for making bispecific antibodyfragments. The fragments comprise a heavy-chain variable domain (V_(H))connected to a light-chain variable domain (V_(L)) by a linker which istoo short to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies may bind to two different epitopes on agiven PRO polypeptide herein. Alternatively, an anti-PRO polypeptide armmay be combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular PRO polypeptide. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aparticular PRO polypeptide. These antibodies possess a PRO-binding armand an arm which binds a cytotoxic agent or a radionuclide chelator,such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody ofinterest binds the PRO polypeptide and further binds tissue factor (TF).

-   -   5. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

-   -   6. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) may beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191–1195(1992) and Shopes, J. Immunol., 148: 2918–2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53:2560–2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3: 219–230 (1989).

-   -   7. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

In another embodiment, the antibody may be conjugated to a “receptor”(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g., avidin) that is conjugatedto a cytotoxic agent (e.g., a radionucleotide).

-   -   8. Immunoliposomes

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286–288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

-   -   9. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO polypeptide identified herein, aswell as other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of various disordersin the form of pharmaceutical compositions.

If the PRO polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA 90:7889–7893 (1993).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

G. Uses for Anti-PRO Antibodies

The anti-PRO antibodies of the invention have various utilities. Forexample, anti-PRO antibodies may be used in diagnostic assays for PRO,e.g., detecting its expression in specific cells, tissues, or serum.Various diagnostic assay techniques known in the art may be used, suchas competitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147–158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al.,J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

Anti-PRO antibodies also are useful for the affinity purification of PROfrom recombinant cell culture or natural sources. In this process, theantibodies against PRO are immobilized on a suitable support, such aSEPHADEX® resin or filter paper, using methods well known in the art.The immobilized antibody then is contacted with a sample containing thePRO to be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO from the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC® accession numbers is the AmericanType Culture Collection, Rockville, Md.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 (Altschul, and Gish, Methods in Enzymology 266: 460–80 (1996); asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a Blast score of 70 (or insome cases 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences. Inaddition, the consensus DNA sequences obtained were often (but notalways) extended using repeated cycles of BLAST and phrap to extend theconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward (.f) and reverse (.r) PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100–1000 bp in length. The probe (.p) sequences are typically40–55 bp in length. In some cases, additional oligonucleotides aresynthesized when the consensus sequence is greater than about 1–1.5 kbp.In order to screen several libraries for a full-length clone, DNA fromthe libraries was screened by PCR amplification, as per Ausubel et al.,Current Protocols in Molecular Biology, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278–1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones Encoding PRO187

A proprietary expressed sequence tag (EST) DNA database (LIFESEQ™,Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST(#843193) was identified which showed homology to fibroblast growthfactor (FGF-8) also known as androgen-induced growth factor. mRNA wasisolated from human fetal lung tissue using reagents and protocols fromInvitrogen, San Diego, Calif. (FAST TRACK™ 2). The cDNA libraries usedto isolate the cDNA clones were constructed by standard methods usingcommercially available reagents (e.g., Invitrogen, San Diego, Calif.,Life Technologies, Gaithersburg, Md.). The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into the cloning vector pRK5D usingreagents and protocols from Life Technologies, Gaithersburg, Md. (SuperScript Plasmid System). The double-stranded cDNA was sized to greaterthan 1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotIcleaved vector. pRK5D is a cloning vector that has an sp6 transcriptioninitiation site followed by an SfiI restriction enzyme site precedingthe XhoI/NotI cDNA cloning sites.

Several libraries from various tissue sources were screened by PCRamplification with the following oligonucleotide probes:

IN843193.f (OLI 315) (SEQ ID NO:3) 5′-CAGTACGTGAGGGACCAGGGCGCCATGA-3′IN843193.r (OLI 317) (SEQ ID NO:4) 5′-CCGGTGACCTGCACGTGCTTGCCA-3′A positive library was then used to isolate clones encoding the PRO187gene using one of the above oligonucleotides and the followingoligonucleotide probe:

IN843193.p (OLI 316) (SEQ ID NO:5)5′-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3′

A cDNA clone was sequenced in entirety. The entire nucleotide sequenceof PRO187 (DNA27864-1155) is shown in FIG. 1 (SEQ ID NO:1). CloneDNA27864-1155 contains a single open reading frame with an apparenttranslational initiation site at nucleotide position 1 (FIG. 1; SEQ IDNO:1). The predicted polypeptide precursor is 205 amino acids long.Clone DNA27864-1155 has been deposited with the ATCC® (designation:DNA27864-1155) and is assigned ATCC® deposit no. ATCC® 209375.

Based on a BLAST and FastA sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence, the PRO187 polypeptideshows 74% amino acid sequence identity (Blast score 310) to humanfibroblast growth factor-8 (androgen-induced growth factor).

Example 3 Use of PRO Polypeptide-Encoding Nucleic Acid as HybridizationProbes

The following method describes use of a nucleotide sequence encoding aPRO polypeptide as a hybridization probe.

DNA comprising the coding sequence of of a PRO polypeptide of interestas disclosed herein may be employed as a probe or used as a basis fromwhich to prepare probes to screen for homologous DNAs (such as thoseencoding naturally-occurring variants of the PRO polypeptide) in humantissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO polypeptide-encoding nucleic acid-derived probe tothe filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO polypeptide can then be identified usingstandard techniques known in the art.

Example 4 Expression of PRO Polypeptides in E. coli

This example illustrates preparation of an unglycosylated form of adesired PRO polypeptide by recombinant expression in E. coli.

The DNA sequence encoding the desired PRO polypeptide is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes forampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the specific PRO polypeptidecoding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO polypeptide can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

PRO187, PRO317, PRO301, PRO224 and PRO238 were successfully expressed inE. coli in a poly-His tagged form, using the following procedure. TheDNA encoding PRO187, PRO317, PRO301, PRO224 or PRO238 was initiallyamplified using selected PCR primers. The primers contained restrictionenzyme sites which correspond to the restriction enzyme sites on theselected expression vector, and other useful sequences providing forefficient and reliable translation initiation, rapid purification on ametal chelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences were then ligated into anexpression vector, which was used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts) clpP(lacIq).Transformants were first grown in LB containing 50 mg/ml carbenicillinat 30° C. with shaking until an O.D.600 of 3–5 was reached. Cultureswere then diluted 50–100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄, 0.71 g sodium citrate 2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20–30 hours at 30° C. with shaking. Samples were removedto verify expression by SDS-PAGE analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets were frozen untilpurification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6–10 g pellets) wasresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1M and 0.02 M, respectively, and the solutionwas stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution wascentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant was diluted with 3–5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. Depending the clarified extract was loaded onto a 5ml Qiagen Ni-NTA metal chelate column equilibrated in the metal chelatecolumn buffer. The column was washed with additional buffer containing50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein waseluted with buffer containing 250 mM imidazole. Fractions containing thedesired protein were pooled and stored at 4° C. Protein concentrationwas estimated by its absorbance at 280 nm using the calculatedextinction coefficient based on its amino acid sequence.

The proteins were refolded by diluting sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes were chosen so that the final protein concentration was between50 to 100 micrograms/ml. The refolding solution was stirred gently at 4°C. for 12–36 hours. The refolding reaction was quenched by the additionof TFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution was filtered through a0.22 micron filter and acetonitrile was added to 2–10% finalconcentration. The refolded protein was chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance were analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein were pooled.Generally, the properly refolded species of most proteins are eluted atthe lowest concentrations of acetonitrile since those species are themost compact with their hydrophobic interiors shielded from interactionwith the reversed phase resin. Aggregated species are usually eluted athigher acetonitrile concentrations. In addition to resolving misfoldedforms of proteins from the desired form, the reversed phase step alsoremoves endotoxin from the samples.

Fractions containing the desired folded PRO187, PRO317, PRO301, PRO224and PRO238 proteins, respectively, were pooled and the acetonitrileremoved using a gentle stream of nitrogen directed at the solution.Proteins were formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodiumchloride and 4% mannitol by dialysis or by gel filtration using G25SUPERFINE™ (Pharmacia) resins equilibrated in the formulation buffer andsterile filtered.

Example 5 Expression of PRO Polypeptides in Mammalian Cells

This example illustrates preparation of a glycosylated form of a desiredPRO polypeptide by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO polypeptide-encoding DNAis ligated into pRK5 with selected restriction enzymes to allowinsertion of the PRO polypeptide DNA using ligation methods such asdescribed in Sambrook et al., supra. The resulting vector is calledpRK5-PRO polypeptide.

In one embodiment, the selected host cells may be 293 cells. Human 293cells (ATCC® CCL 1573) are grown to confluence in tissue culture platesin medium such as DMEM supplemented with fetal calf serum andoptionally, nutrient components and/or antibiotics. About 10 μg pRK5-PROpolypeptide DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543(1982)] and dissolved in 500 μl of 1 mMTris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO polypeptide. The cultures containing transfected cellsmay undergo further incubation (in serum free medium) and the medium istested in selected bioassays.

In an alternative technique, PRO polypeptide may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-PROpolypeptide DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PROpolypeptide can then be concentrated and purified by any selectedmethod, such as dialysis and/or column chromatography.

In another embodiment, PRO polypeptides can be expressed in CHO cells.The pRK5-PRO polypeptide can be transfected into CHO cells using knownreagents such as CaPO₄ or DEAE-dextran. As described above, the cellcultures can be incubated, and the medium replaced with culture medium(alone) or medium containing a radiolabel such as ³⁵S-methionine. Afterdetermining the presence of PRO polypeptide, the culture medium may bereplaced with serum free medium. Preferably, the cultures are incubatedfor about 6 days, and then the conditioned medium is harvested. Themedium containing the expressed PRO polypeptide can then be concentratedand purified by any selected method.

Epitope-tagged PRO polypeptide may also be expressed in host CHO cells.The PRO polypeptide may be subcloned out of the pRK5 vector. Thesubclone insert can undergo PCR to fuse in frame with a selected epitopetag such as a poly-his tag into a Baculovirus expression vector. Thepoly-his tagged PRO polypeptide insert can then be subcloned into a SV40driven vector containing a selection marker such as DHFR for selectionof stable clones. Finally, the CHO cells can be transfected (asdescribed above) with the SV40 driven vector. Labeling may be performed,as described above, to verify expression. The culture medium containingthe expressed poly-His tagged PRO polypeptide can then be concentratedand purified by any selected method, such as by Ni²⁺-chelate affinitychromatography.

PRO211, PRO217, PRO230, PRO219, PRO245, PRO221, PRO258, PRO301, PRO224,PRO222, PRO234, PRO229, PRO223, PRO328 and PRO332 were successfullyexpressed in CHO cells by both a transient and a stable expressionprocedure. In addition, PRO232, PRO265, PRO246, PRO228, PRO227, PRO220,PRO266, PRO269, PRO287, PRO214, PRO231, PRO233, PRO238, PRO244, PRO235,PRO236, PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272,PRO294, PRO295, PRO293, PRO247, PRO303 and PRO268 were successfullytransiently expressed in CHO cells.

Stable expression in CHO cells was performed using the followingprocedure. The proteins were expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins were fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs were subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774–1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA were introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents SUPERFECT® (Quiagen), DOSPER® or FUGENE®(Boehringer Mannheim). The cells were grown and described in Lucas etal., supra. Approximately 3×10⁻⁷ cells are frozen in an ampule forfurther growth and production as described below.

The ampules containing the plasmid DNA were thawed by placement intowater bath and mixed by vortexing. The contents were pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant was aspirated and the cells wereresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells were then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1–2days, the cells were transferred into a 250 mL spinner filled with 150mL selective growth medium and incubated at 37° C. After another 2–3days, a 250 mL, 500 mL and 2000 mL spinners were seeded with 3×10⁵cells/mL. The cell media was exchanged with fresh media bycentrifugation and resuspension in production medium. Although anysuitable CHO media may be employed, a production medium described inU.S. Pat. No. 5,122,469, issued Jun. 16, 1992 was actually used. 3 Lproduction spinner is seeded at 1.2×10⁶ cells/mL. On day 0, the cellnumber pH were determined. On day 1, the spinner was sampled andsparging with filtered air was commenced. On day 2, the spinner wassampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucoseand 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, DowCorning 365 Medical Grade Emulsion). Throughout the production, pH wasadjusted as necessary to keep at around 7.2. After 10 days, or untilviability dropped below 70%, the cell culture was harvested bycentrifugtion and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins were purified using aNi-NTA column (Qiagen). Before purification, imidazole was added to theconditioned media to a concentration of 5 mM. The conditioned media waspumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4–5ml/min. at 4° C. After loading, the column was washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein wassubsequently desalted into a storage buffer containing 10 mM Hepes, 0.14M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of were purified from theconditioned media as follows. The conditioned medium was pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity was assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

PRO211, PRO217, PRO230, PRO232, PRO187, PRO265, PRO219, PRO246, PRO228,PRO533, PRO245, PRO221, PRO227, PRO220, PRO258, PRO266, PRO269, PRO287,PRO214, PRO317, PRO301, PRO224, PRO222, PRO234, PRO231, PRO229, PRO233,PRO238, PRO223, PRO235, PRO236, PRO262, PRO239, PRO257, PRO260, PRO263,PRO270, PRO271, PRO272, PRO294, PRO295, PRO293, PRO247, PRO304, PRO302,PRO307, PRO303, PRO343, PRO328, PRO326, PRO331, PRO332, PRO334, PRO346,PRO268, PRO330, PRO310 and PRO339 were also successfully transientlyexpressed in COS cells.

Example 6 Expression of PRO Polypeptides in Yeast

The following method describes recombinant expression of a desired PROpolypeptide in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO polypeptides from the ADH2/GAPDHpromoter. DNA encoding a desired PRO polypeptide, a selected signalpeptide and the promoter is inserted into suitable restriction enzymesites in the selected plasmid to direct intracellular expression of thePRO polypeptide. For secretion, DNA encoding the PRO polypeptide can becloned into the selected plasmid, together with DNA encoding theADH2/GAPDH promoter, the yeast alpha-factor secretory signal/leadersequence, and linker sequences (if needed) for expression of the PROpolypeptide.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO polypeptide can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing the PRO polypeptide may further be purified usingselected column chromatography resins.

Example 7 Expression of PRO Polypeptides in Baculovirus-Infected InsectCells

The following method describes recombinant expression of PROpolypeptides in Baculovirus-infected insect cells.

The desired PRO polypeptide is fused upstream of an epitope tagcontained with a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thePRO polypeptide or the desired portion of the PRO polypeptide (such asthe sequence encoding the extracellular domain of a transmembraneprotein) is amplified by PCR with primers complementary to the 5′ and 3′regions. The 5′ primer may incorporate flanking (selected) restrictionenzyme sites. The product is then digested with those selectedrestriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BACULOGOLD™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC® CRL 1711) using LIPOFECTIN®(commercially available from GIBCO-BRL). After 4–5 days of incubation at28° C., the released viruses are harvested and used for furtheramplifications. Viral infection and protein expression is performed asdescribed by O'Reilley et al., Baculovirus expression vectors: Alaboratory Manual, Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO polypeptide can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175–179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% Glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO polypeptide are pooled anddialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PROpolypeptide can be performed using known chromatography techniques,including for instance, Protein A or protein G column chromatography.

PRO211, PRO217, PRO230, PRO187, PRO265, PRO246, PRO228, PRO533, PRO245,PRO221, PRO220, PRO258, PRO266, PRO269, PRO287, PRO214, PRO301, PRO224,PRO222, PRO234, PRO231, PRO229, PRO235, PRO239, PRO257, PRO272, PRO294,PRO295, PRO328, PRO326, PRO331, PRO334, PRO346 and PRO310 weresuccessfully expressed in baculovirus infected Sf9 or high5 insectcells. While the expression was actually performed in a 0.5–2 L scale,it can be readily scaled up for larger (e.g. 8 L) preparations. Theproteins were expressed as an IgG construct (immunoadhesin), in whichthe protein extracellular region was fused to an IgG1 constant regionsequence containing the hinge, CH2 and CH3 domains and/or in poly-Histagged forms.

Following PCR amplification, the respective coding sequences weresubcloned into a baculovirus expression vector (pb.PH.IgG for IgGfusions and pb.PH.His.c for poly-His tagged proteins), and the vectorand BACULOGOLD™ baculovirus DNA (Pharmingen Corp., San Diego, Calif.)were co-transfected into 105 Spodoptera frugiperda (“Sf9”) cells (ATCC®CRL 1711), using LIPOFECTIN® (Gibco BRL). pb.PH.IgG and pb.PH.His aremodifications of the commercially available baculovirus expressionvector pVL1393 (Pharmingen), with modified polylinker regions to includethe His or Fc tag sequences. The cells were grown in Hink's TNM-FHmedium supplemented with 10% FBS (HYCLONE® (Hyclone Laboratories, Inc.,Logan, Utah). Cells were incubated for 5 days at 28° C. The supernatantwas harvested and subsequently used for the first viral amplification byinfecting Sf9 cells in Hink's TNM-FH medium supplemented with 10% FBS atan approximate multiplicity of infection (MOI) of 10. Cells wereincubated for 3 days at 28° C. The supernatant was harvested and theexpression of the constructs in the baculovirus expression vector wasdetermined by batch binding of 1 ml of supernatant to 25 mL of Ni-NTAbeads (QIAGEN) for histidine tagged proteins or PROTEIN A-SEPHAROSE™CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGEanalysis comparing to a known concentration of protein standard byCoomassie blue staining.

The first viral amplification supernatant was used to infect a spinnerculture (500 ml) of Sf9 cells grown in ESF-921 medium (ExpressionSystems LLC) at an approximate MOI of 0.1. Cells were incubated for 3days at 28° C. The supernatant was harvested and filtered. Batch bindingand SDS-PAGE analysis was repeated, as necessary, until expression ofthe spinner culture was confirmed.

The conditioned medium from the transfected cells (0.5 to 3 L) washarvested by centrifugation to remove the cells and filtered through0.22 micron filters. For the poly-His tagged constructs, the proteinconstruct were purified using a Ni-NTA column (Qiagen). Beforepurification, imidazole was added to the conditioned media to aconcentration of 5 mM. The conditioned media were pumped onto a 6 mlNi-NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3M NaCl and 5 mM imidazole at a flow rate of 4–5 ml/min. at 4° C. Afterloading, the column was washed with additional equilibration buffer andthe protein eluted with equilibration buffer containing 0.25 Mimidazole. The highly purified protein was subsequently desalted into astorage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at −80° C.

Immunoadhesin (Fc containing) constructs of proteins were purified fromthe conditioned media as follows. The conditioned media were pumped ontoa 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mMNa phosphate buffer, pH 6.8. After loading, the column was washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein was immediately neutralized bycollecting 1 ml fractions into tubes containing 275 mL of 1 M Trisbuffer, pH 9. The highly purified protein was subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity of the proteins was verified by SDS polyacrylamide gel (PEG)electrophoresis and N-terminal amino acid sequencing by Edmandegradation.

Example 8 Preparation of Antibodies that Bind to PRO Polypeptides

This example illustrates preparation of monoclonal antibodies which canspecifically bind to a PRO polypeptide.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO polypeptide, fusion proteins containingthe PRO polypeptide, and cells expressing recombinant PRO polypeptide onthe cell surface. Selection of the immunogen can be made by the skilledartisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO polypeptide immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1–100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO polypeptide antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO polypeptide. Three to four days later, the mice aresacrificed and the spleen cells are harvested. The spleen cells are thenfused (using 35% polyethylene glycol) to a selected murine myeloma cellline such as P3X63AgU.1, available from ATCC®, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstthe PRO polypeptide. Determination of “positive” hybridoma cellssecreting the desired monoclonal antibodies against the PRO polypeptideis within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PROpolypeptide monoclonal antibodies. Alternatively, the hybridoma cellscan be grown in tissue culture flasks or roller bottles. Purification ofthe monoclonal antibodies produced in the ascites can be accomplishedusing ammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 9 Chimeric PRO Polypeptides

PRO polypeptides may be expressed as chimeric proteins with one or moreadditional polypeptide domains added to facilitate protein purification.Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS™ extension/affinity purification system (Immunex Corp.,Seattle Wash.). The inclusion of a cleavable linker sequence such asFactor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and the PRO polypeptide sequence may be useful tofacilitate expression of DNA encoding the PRO polypeptide.

Example 10 Purification of PRO Polypeptides Using Specific Antibodies

Native or recombinant PRO polypeptides may be purified by a variety ofstandard techniques in the art of protein purification. For example,pro-PRO polypeptide, mature PRO polypeptide, or pre-PRO polypeptide ispurified by immunoaffinity chromatography using antibodies specific forthe PRO polypeptide of interest. In general, an immunoaffinity column isconstructed by covalently coupling the anti-PRO polypeptide antibody toan activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PROpolypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

A soluble PRO polypeptide-containing preparation is passed over theimmunoaffinity column, and the column is washed under conditions thatallow the preferential absorbance of PRO polypeptide (e.g., high ionicstrength buffers in the presence of detergent). Then, the column iseluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2–3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 11 Drug Screening

This invention is particularly useful for screening compounds by usingPRO polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO polypeptide or fragment employed in such atest may either be free in solution, affixed to a solid support, borneon a cell surface, or located intracellularly. One method of drugscreening utilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO polypeptide-associated diseaseor disorder. These methods comprise contacting such an agent with an PROpolypeptide or fragment thereof and assaying (I) for the presence of acomplex between the agent and the PRO polypeptide or fragment, or (ii)for the presence of a complex between the PRO polypeptide or fragmentand the cell, by methods well known in the art. In such competitivebinding assays, the PRO polypeptide or fragment is typically labeled.After suitable incubation, free PRO polypeptide or fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind to PROpolypeptide or to interfere with the PRO polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO polypeptide, the peptide test compounds arereacted with PRO polypeptide and washed. Bound PRO polypeptide isdetected by methods well known in the art. Purified PRO polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. In addition, non-neutralizing antibodies can beused to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 12 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO polypeptide) orof small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c.f., Hodgson, Bio/Technology, 9: 19–21 (1991)).

In one approach, the three-dimensional structure of the PRO polypeptide,or of an PRO polypeptide-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry, 31:7796–7801(1992) or which act as inhibitors, agonists, or antagonists of nativepeptides as shown by Athauda et al., J. Biochem., 113:742–746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition tox-ray crystallography.

Example 13 Ability of PRO Polypeptides to Inhibit Vascular EndothelialGrowth Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth(Assay 9)

The ability of various PRO polypeptides to inhibit VEGF stimulatedproliferation of endothelial cells was tested. Polypeptides testingpositive in this assay are useful for inhibiting endothelial cell growthin mammals where such an effect would be beneficial, e.g., forinhibiting tumor growth.

Specifically, bovine adrenal cortical capillary endothelial cells (ACE)(from primary culture, maximum of 12–14 passages) were plated in 96-wellplates at 500 cells/well per 100 microliter. Assay media included lowglucose DMEM, 10% calf serum, 2 mM glutamine, and 1×penicillin/streptomycin/fungizone. Control wells included the following:(1) no ACE cells added; (2) ACE cells alone; (3) ACE cells plus 5 ng/mlFGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE cells plus 3 ng/ml VEGFplus 1 ng/ml TGF-beta; and (6) ACE cells plus 3 ng/ml VEGF plus 5 ng/mlLIF. The test samples, poly-his tagged PRO polypeptides (in 100microliter volumes), were then added to the wells (at dilutions of 1%,0.1% and 0.01%, respectively). The cell cultures were incubated for 6–7days at 37° C./5% CO₂. After the incubation, the media in the wells wasaspirated, and the cells were washed 1× with PBS. An acid phosphatasereaction mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1%Triton X-100, 10 mM p-nitrophenyl phosphate) was then added to eachwell. After a 2 hour incubation at 37° C., the reaction was stopped byaddition of 10 microliters 1N NaOH. Optical density (OD) was measured ona microplate reader at 405 nm.

The activity of PRO polypeptides was calculated as the percentinhibition of VEGF (3 ng/ml) stimulated proliferation (as determined bymeasuring acid phosphatase activity at OD 405 nm) relative to the cellswithout stimulation. TGF-beta was employed as an activity reference at 1ng/ml, since TGF-beta blocks 70–90% of VEGF-stimulated ACE cellproliferation. The results are indicative of the utility of the PROpolypeptides in cancer therapy and specifically in inhibiting tumorangiogenesis. Numerical values (relative inhibition) are determined bycalculating the percent inhibition of VEGF stimulated proliferation bythe PRO polypeptides relative to cells without stimulation and thendividing that percentage into the percent inhibition obtained by TGF-βat 1 ng/ml which is known to block 70–90% of VEGF stimulated cellproliferation. The results are considered positive if the PROpolypeptide exhibits 30% or greater inhibition of VEGF stimulation ofendothelial cell growth (relative inhibition 30% or greater).

The following polypeptides tested positive in this assay: PRO211,PRO217, PRO187, PRO219, PRO246, PRO228, PRO245, PRO221, PRO258, PRO301,PRO224, PRO272, PRO328, PRO331, PRO224, PRO328, PRO272, PRO301, PRO331and PRO214.

Example 14 Detection of Polypeptides that Affect Glucose and/or FFAUptake in Skeletal Muscle (Assay 106)

This assay is designed to determine whether PRO polypeptides show theability to affect glucose or FFA uptake by skeletal muscle cells. PROpolypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by skeletal muscle would bebeneficial including, for example, diabetes or hyper- orhypo-insulinemia.

In a 96 well format, PRO polypeptides to be assayed are added to primaryrat differentiated skeletal muscle, and allowed to incubate overnight.Then fresh media with the PRO polypeptide and +/−insulin are added tothe wells. The sample media is then monitored to determine glucose andFFA uptake by the skeletal muscle cells. The insulin will stimulateglucose and FFA uptake by the skeletal muscle, and insulin in mediawithout the PRO polypeptide is used as a positive control, and a limitfor scoring. As the PRO polypeptide being tested may either stimulate orinhibit glucose and FFA uptake, results are scored as positive in theassay if greater than 1.5 times or less than 0.5 times the insulincontrol.

The following PRO polypeptides tested positive as either stimulators orinhibitors of glucose and/or FFA uptake in this assay: PRO187, PRO211,PRO221, PRO222, PRO224, PRO230, PRO239, PRO231, PRO245, PRO247, PRO258,PRO269, PRO328 and PRO533.

Example 15 Gene Amplification

This example shows that certain PRO polypeptide-encoding genes areamplified in the genome of certain human lung, colon and/or breastcancers and/or cell lines. Amplification is associated withoverexpression of the gene product, indicating that the polypeptides areuseful targets for therapeutic intervention in certain cancers such ascolon, lung, breast and other cancers and diagnostic determination ofthe presence of those cancers. Therapeutic agents may take the form ofantagonists of the PRO polypeptide, for example, murine-human chimeric,humanized or human antibodies against a PRO polypeptide.

The starting material for the screen was genomic DNA isolated from avariety cancers. The DNA is quantitated precisely, e.g.,fluorometrically. As a negative control, DNA was isolated from the cellsof ten normal healthy individuals which was pooled and used as assaycontrols for the gene copy in healthy individuals (not shown). The 5′nuclease assay (for example, TAQMAN™) and real-time quantitative PCR(for example, ABI PRIZM 7700 SEQUENCE DETECTION SYSTEM™ (Perkin Elmer,Applied Biosystems Division, Foster City, Calif.)), were used to findgenes potentially amplified in certain cancers. The results were used todetermine whether the DNA encoding the PRO polypeptide isover-represented in any of the primary lung or colon cancers or cancercell lines or breast cancer cell lines that were screened. The primarylung cancers were obtained from individuals with tumors of the type andstage as indicated in Table 7. An explanation of the abbreviations usedfor the designation of the primary tumors listed in Table 7 and theprimary tumors and cell lines referred to throughout this example aregiven below.

The results of the TAQMAN™ are reported in delta (Δ) Ct units. One unitcorresponds to 1 PCR cycle or approximately a 2-fold amplificationrelative to normal, two units corresponds to 4-fold, 3 units to 8-foldamplification and so on. Quantitation was obtained using primers and aTAQMAN™ fluorescent probe derived from the PRO polypeptide-encodinggene. Regions of the PRO polypeptide-encoding gene which are most likelyto contain unique nucleic acid sequences and which are least likely tohave spliced out introns are preferred for the primer and probederivation, e.g., 3′-untranslated regions. The sequences for the primersand probes (forward, reverse and probe) used for the PRO polypeptidegene amplification analysis were as follows:

PRO187 (DNA27864-1155) 27864.tm.p: 5′-GCAGATTTTGAGGACAGCCACCTCCA-3′ (SEQID NO:6) 27864.tm.f: 5′-GGCCTTGCAGACAACCGT-3′ (SEQ ID NO:7) 27864.tm.r:5′-CAGACTGAGGGAGATCCGAGA-3′ (SEQ ID NO:8) 27864.tm.p2:5′-CAGCTGCCCTTCCCCAACCA-3′ (SEQ ID NO:9) 27864.tm.f2:5′-CATCAAGCGCCTCTACCA-3′ (SEQ ID NO:10) 27864.tm.r2:5′-CACAAACTCGAACTGCTTCTG-3′ (SEQ ID NO:11)

The 5′ nuclease assay reaction is a fluorescent PCR-based techniquewhich makes use of the 5′ exonuclease activity of Taq DNA polymeraseenzyme to monitor amplification in real time. Two oligonucleotideprimers (forward [.f] and reverse [.r]) are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe (.p), isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI PRIZM 7700 SEQUENCE DETECTION SYSTEM™. The systemconsists of a thermocycler, laser, charge-coupled device (CCD) cameraand computer. The system amplifies samples in a 96-well format on athermocycler. During amplification, laser-induced fluorescent signal iscollected in real-time through fiber optics cables for all 96 wells, anddetected at the CCD. The system includes software for running theinstrument and for analyzing the data.

5′ Nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer DNA results to normal human DNA results.

Table 7 describes the stage, T stage and N stage of various primarytumors which were used to screen the PRO polypeptide compounds of theinvention.

TABLE 7 Primary Lung and Colon Tumor Profiles Other Dukes T N PrimaryTumor Stage Stage Stage Stage Stage Stage Human lung tumor AdenoCa(SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa (SRCC725) [LT1a] IIB T3N0 Human lung tumor AdenoCa (SRCC726) [LT2] IB T2 N0 Human lung tumorAdenoCa (SRCC727) [LT3] IIIA T1 N2 Human lung tumor AdenoCa (SRCC728)[LT4] IB T2 N0 Human lung tumor SqCCa (SRCC729) [LT6] IB T2 N0 Humanlung tumor Aden/SqCCa (SRCC730) [LT7] IA T1 N0 Human lung tumor AdenoCa(SRCC731) [LT9] IB T2 N0 Human lung tumor SqCCa (SRCC732) [LT10] IIB T2N1 Human lung tumor SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumorAdenoCa (SRCC734) [LT12] IV T2 N0 Human lung tumor AdenoSqCCa(SRCC735)[LT13] IB T2 N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2N0 Human lung tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumorSqCCa (SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18]IB T2 N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human lungtumor LCCa (SRCC741) [LT21] IIB T3 N1 Human lung AdenoCa (SRCC811)[LT22] 1A T1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D pT4 N0 Humancolon AdenoCa (SRCC743) [CT3] B pT3 N0 Human colon AdenoCa (SRCC744)[CT8] B T3 N0 Human colon AdenoCa (SRCC745) [CT10] A pT2 N0 Human colonAdenoCa (SRCC746) [CT12] MO, R1 B T3 N0 Human colon AdenoCa (SRCC747)[CT14] pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] M1, R2 DT4 N2 Human colon AdenoCa (SRCC749) [CT16] pMO B pT3 pN0 Human colonAdenoCa (SRCC750) [CT17] C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1]MO, R1 B pT3 N0 Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colonAdenoCa (SRCC753) [CT5] G2 C1 pT3 pN0 Human colon AdenoCa (SRCC754)[CT6] pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon AdenoCa(SRCC757) [CT11] B T3 N0 Human colon AdenoCa (SRCC758) [CT18] MO, RO BpT3 pN0DNA Preparation:

DNA was prepared from cultured cell lines, primary tumors, normal humanblood. The isolation was performed using purification kit, buffer setand protease and all from Quiagen, according to the manufacturer'sinstructions and the description below.

Cell Culture Lysis:

Cells were washed and trypsinized at a concentration of 7.5×10⁸ per tipand pelleted by centrifuging at 1000 rpm for 5 minutes at 4° C.,followed by washing again with ½ volume of PBS recentrifugation. Thepellets were washed a third time, the suspended cells collected andwashed 2× with PBS. The cells were then suspended into 10 ml PBS. BufferC1 was equilibrated at 4° C. Qiagen protease #19155 was diluted into6.25 ml cold ddH₂ 0 to a final concentration of 20 mg/ml andequilibrated at 4° C. 10 ml of G2 Buffer was prepared by diluting QiagenRNAse A stock (100 mg/ml) to a final concentration of 200 μg/ml.

Buffer C1 (10 ml, 4° C.) and ddH2O (40 ml, 4° C.) were then added to the10 ml of cell suspension, mixed by inverting and incubated on ice for 10minutes. The cell nuclei were pelleted by centrifuging in a Beckmanswinging bucket rotor at 2500 rpm at 4° C. for 15 minutes. Thesupernatant was discarded and the nuclei were suspended with a vortexinto 2 ml Buffer C1 (at 4° C.) and 6 ml ddH₂O, followed by a second 4°C. centrifugation at 2500 rpm for 15 minutes. The nuclei were thenresuspended into the residual buffer using 200 μl per tip. G2 buffer (10ml) was added to the suspended nuclei while gentle vortexing wasapplied. Upon completion of buffer addition, vigorous vortexing wasapplied for 30 seconds. Quiagen protease (200 μl, prepared as indicatedabove) was added and incubated at 50° C. for 60 minutes. The incubationand centrifugation was repeated until the lysates were clear (e.g.,incubating additional 30–60 minutes, pelleting at 3000×g for 10 min., 4°C.).

Solid Human Tumor Sample Preparation and Lysis:

Tumor samples were weighed and placed into 50 ml conical tubes and heldon ice. Processing was limited to no more than 250 mg tissue perpreparation (1 tip/preparation). The protease solution was freshlyprepared by diluting into 6.25 ml cold ddH₂O to a final concentration of20 mg/ml and stored at 4° C. G2 buffer (20 ml) was prepared by dilutingDNAse A to a final concentration of 200 mg/ml (from 100 mg/ml stock).The tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds usingthe large tip of the polytron in a laminar-flow TC hood in order toavoid inhalation of aerosols, and held at room temperature. Betweensamples, the polytron was cleaned by spinning at 2×30 seconds each in 2LddH₂0, followed by G2 buffer (50 ml). If tissue was still present on thegenerator tip, the apparatus was disassembled and cleaned.

Quiagen protease (prepared as indicated above, 1.0 ml) was added,followed by vortexing and incubation at 50° C. for 3 hours. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30–60 minutes, pelleting at 3000×g for 10min., 4° C.).

Human Blood Preparation and Lysis:

Blood was drawn from healthy volunteers using standard infectious agentprotocols and citrated into 10 ml samples per tip. Quiagen protease wasfreshly prepared by dilution into 6.25 ml cold ddH₂O to a finalconcentration of 20 mg/ml and stored at 4° C. G2 buffer was prepared bydiluting RNAse A to a final concentration of 200 μg/ml from 100 mg/mlstock. The blood (10 ml) was placed into a 50 ml conical tube and 10 mlC1 buffer and 30 ml ddH₂O (both previously equilibrated to 4° C.) wereadded, and the components mixed by inverting and held on ice for 10minutes. The nuclei were pelleted with a Beckman swinging bucket rotorat 2500 rpm, 4° C. for 15 minutes and the supernatant discarded. With avortex, the nuclei were suspended into 2 ml C1 buffer (4° C.) and 6 mlddH₂O (4° C.). Vortexing was repeated until the pellet was white. Thenuclei were then suspended into the residual buffer using a 200 μl tip.G2 buffer (10 ml) were added to the suspended nuclei while gentlyvortexing, followed by vigorous vortexing for 30 seconds. Quiagenprotease was added (200 μl) and incubated at 50° C. for 60 minutes. Theincubation and centrifugation was repeated until the lysates were clear(e.g., incubating additional 30–60 minutes, pelleting at 3000×g for 10min., 4° C.).

Purification of Cleared Lysates:

(1) Isolation of Genomic DNA:

Genomic DNA was equilibrated (1 sample per maxi tip preparation) with 10ml QBT buffer. QF elution buffer was equilibrated at 50° C. The sampleswere vortexed for 30 seconds, then loaded onto equilibrated tips anddrained by gravity. The tips were washed with 2×15 ml QC buffer. The DNAwas eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15 mlQF buffer (50° C.). Isopropanol (10.5 ml) was added to each sample, thetubes covered with parafin and mixed by repeated inversion until the DNAprecipitated. Samples were pelleted by centrifugation in the SS-34 rotorat 15,000 rpm for 10 minutes at 4° C. The pellet location was marked,the supernatant discarded, and 10 ml 70% ethanol (4° C.) was added.Samples were pelleted again by centrifugation on the SS-34 rotor at10,000 rpm for 10 minutes at 4° C. The pellet location was marked andthe supernatant discarded. The tubes were then placed on their side in adrying rack and dried 10 minutes at 37° C., taking care not to overdrythe samples.

After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) andplaced at 50° C. for 1–2 hours. Samples were held overnight at 4° C. asdissolution continued. The DNA solution was then transferred to 1.5 mltubes with a 26 gauge needle on a tuberculin syringe. The transfer wasrepeated 5× in order to shear the DNA. Samples were then placed at 50°C. for 1–2 hours.

(2) Quantitation of Genomic DNA and Preparation for Gene AmplificationAssay:

The DNA levels in each tube were quantified by standard A₂₆₀, A₂₈₀spectrophotometry on a 1:20 dilution (5 μl DNA+95 μl ddH₂O) using the0.1 ml quartz cuvetts in the Beckman DU640 spectrophotometer. A₂₆₀/A₂₈₀ratios were in the range of 1.8–1.9. Each DNA samples was then dilutedfurther to approximately 200 ng/ml in TE (pH 8.5). If the originalmaterial was highly concentrated (about 700 ng/μl), the material wasplaced at 50° C. for several hours until resuspended.

Fluorometric DNA quantitation was then performed on the diluted material(20–600 ng/ml) using the manufacturer's guidelines as modified below.This was accomplished by allowing a Hoeffer DyNA Quant 200 fluorometerto warm-up for about 15 minutes. The Hoechst dye working solution(#H33258, 10 μl, prepated within 12 hours of use) was diluted into 100ml 1×TNE buffer. A 2 ml cuvette was filled with the fluorometersolution, placed into the machine, and the machine was zeroed. pGEM3Zf(+) (2 μl, lot #360851026) was added to 2 ml of fluorometer solutionand calibrated at 200 units. An additional 2 μl of pGEM 3Zf(+) DNA wasthen tested and the reading confirmed at 400+/−10 units. Each sample wasthen read at least in triplicate. When 3 samples were found to be within10% of each other, their average was taken and this value was used asthe quantification value.

The fluorometricly determined concentration was then used to dilute eachsample to 10 ng/μl in ddH₂O. This was done simultaneously on alltemplate samples for single TAQMAN™ plate assay, and with enoughmaterial to run 500–1000 assays. The samples were tested in triplicatewith TAQMAN™ primers and probe both B-actin and GAPDH on a single platewith normal human DNA and no-template controls. The diluted samples wereused provided that the CT value of normal human DNA subtracted from testDNA was +/−1 Ct. The diluted, lot-qualified genomic DNA was stored in1.0 ml aliquots at −80° C. Aliquots which were subsequently to be usedin the gene amplification assay were stored at 4° C. Each 1 ml aliquotis enough for 8–9 plates or 64 tests.

Gene Amplification Assay:

The PRO polypeptide compounds of the invention were screened in thefollowing primary tumors and the resulting ΔCt values greater than orequal to 1.0 are reported in Table 8 below.

TABLE 8 ΔCt values in lung and colon primary tumors and cell line modelsPrimary Tumors or Cell lines PRO187 PRO533 PRO214 PRO343 PRO211 PRO230LT7 LT13 2.74 1.85 2.71 1.88 3.42 2.98 1.83 2.23 2.26 3.22 2.44 2.842.15 2.75 2.53 1.82 LT3 1.57 1.97 LT4 1.17 LT9 1.42 LT12 2.70 1.38 2.231.51 2.86 2.90 1.49 1.50 1.27 2.96 2.27 2.92 1.25 2.68 2.28 1.34 LT301.67 2.13 1.36 LT21 1.26 1.09 LT1-a 1.02 1.18 LT6 LT10 1.96 LT11 1.091.67 1.00 2.05 1.32 1.80 1.89 1.14 1.54 LT15 3.75 1.77 3.62 2.44 4.323.92 1.58 1.30 2.16 4.47 3.49 3.64 2.94 3.56 3.32 2.68 LT16 2.10 1.661.70 1.25 1.15 2.04 1.83 LT17 1.32 1.93 1.15 1.85 1.26 1.87 2.30 1.391.30 1.33 1.30 LT18 1.17 LT19 4.05 1.67 2.09 3.82 2.42 4.05 3.99 1.982.55 4.92 4.93 3.78 4.76 HF-000840 1.58 Calu-1 1.08 SW900 1.86 CT2 3.562.49 1.95 1.42 3.49 CT3 2.06 1.15 1.34 CT8 1.01 1.48 1.29 1.58 CT10 1.811.84 1.88 1.00 1.49 CT12 1.81 1.74 1.13 CT14 1.82 2.48 2.33 1.72 CT151.63 2.06 1.41 CT16 1.95 1.78 1.40 CT17 2.04 2.40 1.74 CT1 1.24 1.221.27 1.25 1.34 1.46 1.14 CT4 1.36 1.77 1.33 1.32 1.42 1.02 CT5 2.96 1.562.68 1.76 2.27 2.99 2.76 1.64 CT6 1.10 1.33 1.01 1.14 CT7 1.40 1.66 1.39CT9 1.39 1.16 CT11 2.22 2.05 1.55 2.01 1.75 2.26 1.85 1.83 HF000539 1.57SW620 1.14 HF000611 4.64 HF000733 1.93 2.33 HF000716 1.68 2.82 CT18Primary Tumors or Cell lines PRO246 PRO317 PRO232 PRO269 PRO304 PRO339LT7 1.52 1.04 1.08 LT13 1.63 1.90 1.27 1.29 1.04 1.68 2.24 2.93 LT3 1.061.86 1.17 LT4 1.18 LT9 1.04 1.80 1.03 LT12 1.54 2.54 2.40 1.14 1.15 1.262.47 1.74 LT30 LT21 1.50 LT1-a 1.29 LT6 1.93 LT10 1.07 2.57 LT11 3.432.20 1.14 1.51 1.39 1.41 2.33 1.02 LT15 2.11 2.06 1.86 1.36 1.34 1.562.76 1.63 LT16 1.55 1.00 1.08 1.33 LT17 2.68 2.29 1.35 1.42 1.68 1.631.69 2.03 1.10 LT18 1.04 LT19 1.91 2.51 1.21 1.60 1.15 1.68 2.03 1.16HF-000840 Calu-1 SW900 CT2 2.75 2.36 CT3 CT8 CT10 1.88 1.55 CT12 CT141.36 1.24 CT15 1.33 1.04 CT16 CT17 CT1 2.41 CT4 1.10 1.17 2.05 CT5 1.331.59 2.39 CT6 CT7 1.00 CT9 1.09 1.24 1.13 CT11 1.48 1.92 1.12 HF000539SW620 HF000611 HF000733 HF000716 CT18 1.29

Because amplification of the various DNA's as described above occurs invarious tumors, it is likely associated with tumor formation and/orgrowth. As a result, antagonists (e.g., antibodies) directed againstthese polypeptides would be expected to be useful in cancer therapy.

Example 17 Tissue Expression Distribution

Oligonucleotide probes were constructed from some of the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200–600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human adult and/or fetal tissuesources and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tissues tested.Knowledge of the expression pattern or the differential expression ofthe PRO polypeptide-encoding nucleic acid in various different humantissue types provides a diagnostic marker useful for tissue typing, withor without other tissue-specific markers, for determining the primarytissue source of a metastatic tumor, and the like. These assays providedthe following results.

Tissues With Tissues Lacking DNA Molecule Significant ExpressionSignificant Expression DNA34436-1238 lung, placenta, brain testisDNA35557-1137 lung, kidney, brain placenta DNA35599-1168 kidney, brainliver, placenta DNA35668-1171 liver, lung, kidney placenta, brainDNA36992-1168 liver, lung, kidney, brain placenta DNA39423-1182 kidney,brain liver DNA40603-1232 liver brain, kidney, lung DNA40604-1187 liverbrain, kidney, lung DNA41379-1236 lung, brain liver DNA33206-1165 heart,spleen, dendrocytes substantia nigra, hippocampus, cartilage, prostate,HUVEC DNA34431-1177 spleen, HUVEC, cartilage, brain, colon tumor, heart,uterus prostate, THP-1 macrophages DNA41225-1217 HUVEC, uterus, colonspleen, brain, heart, tumor, cartilage, prostate IM-9 lymphoblastsDeposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. USA(ATCC®):

-   Material ATCC® Dep. No. Deposit Date-   DNA27864-1155 ATCC® 209375 Oct. 16, 1997

These deposit were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCC®under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC®, which assures that all restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of thepertinent U.S. patent, assures permanent and unrestricted availabilityof the progeny of the culture of the deposit to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An isolated polypeptide having at least 99% amino acid sequenceidentity to: (a) the amino acid sequence of the polypeptide of SEQ IDNO: 2; (b) the amino acid sequence of the polypeptide of SEQ ID NO: 2,lacking its associated signal peptide; or (c) the amino acid sequence ofthe polypeptide encoded by the full-length coding sequence of the cDNAdeposited under ATCC accession number 209375, wherein said polypeptideinhibits endothelial cell growth.
 2. A chimeric polypeptide comprising apolypeptide according to claim 1 fused to a heterologous polypeptide. 3.The chimeric polypeptide of claim 2, wherein said heterologouspolypeptide is an epitope tag or an Fc region of an immunoglobulin. 4.An isolated polypeptide comprising: (a) the amino acid sequence of thepolypeptide of SEQ ID NO:2; (b) the amino acid sequence of thepolypeptide of SEQ ID NO:2, lacking its associated signal peptide; or(c) the amino acid sequence of the polypeptide encoded by thefull-length coding sequence of the cDNA deposited under ATCC accessionnumber 209375.